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[84] Animal systems synthesizing mucopolysaccharides
[84]
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCH&RIDES
491
o-amino acids a water-soluble product is formed. 1 These polymers spread on application to filter paper and are referred to as "spreading products." They yield only the monomer, GlcNAc-MurNAc(.L-ala.D-glu.mesoDAP.D-ala-D-ala) on hydrolysis with lysozyme. The presence of 1 or 2 D-alanine residues in such products is measured by the use of doubly labeled substrate containing 3H-meso-DAP and 14C-D-ala.14C-D-ala. The linear products synthesized in whole cells treated with penicillin or with D-amino acids have been seen by electron microscopy as linear strands/, s 7 C. Lark, D. Bradley, and K. G. Lark, Biochim. Biophys. Acta 78, 278 (1963). s p. Fitz-James and R. Hancock, J. Cell Biol. 26, 657 (1965).
[84] Animal Systems Synthesizing Mucopolysaccharides By JEREMIAH E. SILBERT I. Chondroitin UDP-N-acetylgalactosamine + UDP-glucuronic acid --* chondroitin Assay Method
Principle. A microsomal preparation from chick embryo cartilage catalyzes the incorporation of N-acetyl-3H galactosamine and glucuronic acid-14C from UDP-N-acetyl-~H galactosamine and UDP-glucuronic acid-14C into a polysaccharide having the characteristics of chondroitin? Enzyme activity is assayed by isolation and counting of the radioactive polysaccharide. Reagents UDP-N-acetyl-3H galactosamine ( ~ 2 0 X 106 cpm/micromole) ~ UDP-glucuronic acidJ4C ( ~ 1 0 X 106 cpm/micromole) 2 Tris, 0.5 M, pH 7.8 MgC12, 0.1 M Sucrose, 2 M Pancreatin, 1%, in 0.05 M Tris, pH 9.0
Procedure. ENZYMATIC REACTION.The reaction mixture is prepared as follows in a volume made up to 0.025 ml: Tris (pH 7.8), 5 t~l; MgCI~, 5 ~1; sucrose, 3 t~l; UDP-N-acetyl-~H galactosamine, 0.025 micromoles (5 X 105 epm) ; UDP-glucuronic acid-~4C, 0.025 micromoles (2.5 X 105 cpm). The reaction is started by the addition of 0.025 ml of enzyme prep1j. E. Silbert, J. Biol. Chem. 239, 1310 (1964). J. E. Silbert, Biochem. Biophys. Res. Commun. 9, 266 (1962).
492
ENZYMES OF C O M P L E X SACCHARIDE SYNTHESIS
[84]
aration containing about 0.05-0.07 mg of protein. After incubation at 37 ° for 90 minutes the reaction is stopped by placing the tubes containing the reaction mixtures into a boiling water bath for 2 minutes. ISOLATION OF THE RADIOACTIVE POLYSACCHARIDE. The total reaction mixture, including all the particulate material, is streaked in a 3-cm band on Whatman No. 1 chromatography paper. The ehromatograms are then developed overnight in a descending system, utilizing 95% ethanol-1 M ammonium acetate (5:2).3 All the radioactive sugar nucleotides and degradation products move varying distances down the paper, while the radioactive polysaccharide remains at the origin. The chromatogram is then dried, and the paper at the origin is cut out and incubated overnight at 37 ° in 5 ml of the pancreatin solution. This solubilizes protein-bound radioactive polysaccharide. An aliquot of the pancreatin digest is then dialyzed at room temperature for 24 hours against water with three changes of water. The volume of the liquid within the dialysis tubing is measured, and the liquid is then centrifuged at 20,000 g for 20 minutes. The clear supernatant contains the radioactive polysaccharide. The radioactive material can be identified as a macromolecule by gel filtration.1 I t can be identified as chondroitin by acid hydrolysis and identification of the sugar constituents, chromatography on DEAEcellulose, precipitation with cetyltrimethylammonium bromide, and susceptibility to degradation by various hyaluronidases.1 DETERMINATION OF THE RADIOACTIVITY.An aliquot of the clear supernatant is added to 15 ml of scintillation medium.4 Both 14C and 3H are determined simultaneously in a dual channel scintillation spectrometer. Alternatively, the dialysis step may be omitted, since all the radioactivity solubilized from the origin of the chromatogram remains within the dialysis tubing. However, the dialyzed preparation gives a somewhat more reliable assay because of quenching variability in counting of nondialyzed preparations. Purification Procedure Fertilized eggs are incubated at 37 ° in an incubator until the embryos are 13-15 days old. Each embryo is removed from its egg and its limbs are disarticulated from the pelvis. The muscle is stripped off, exposing both ends of each femur and tibia. The end cartilage at the joint surfaces is cut away with a fine scissors or scalpel blade, and the highly cellular epiphyses are expressed onto a glass plate by gentle ~A. C. Paladini and L. F. Leloir, Biochem. J. 51, 426 (1952). G. A. Bray, Anal. Biochem. I, 279 (1960).
[84]
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCHARIDES
493
pressure on the shaft of the bones 2-3 mm from the cut ends. The epiphyses are placed immediately in 5-10 ml of 0.25 M sucrose in an ice bath. Ordinarily, the time involved for processing 3{)-40 embryos has not exceeded 3 hours. The epiphyses are then homogenized in the sucrose solution at ()-4 ° with a Potter-Elvehjem glass homogenizer. The remainder of the enzyme preparation is carried out at {)-4° . The viscous homogenate is centrifuged at 10,000 g for l0 minutes, and the supernatant is then centrifuged at 100,000 g for 20 minutes. After centrifugation, the supernatant is removed, the pellet is resuspended in 0.25 M sucrose, and the preparation is centrifuged again at 100,000 g. The supernatant is removed and the final pellet (usually about 0.1 ml) is suspended in 0.25 ml of 0.25 M sucrose. Properties The incorporation of N-aeetylgalactosamine and glucuronic acid catalyzed by the microsomal preparation is equimolar, and the presence of both UDP-N-acetylgalactosamine and UDP-glucuronie acid is necessary for maximal formation. However, UDP-N-acetylglucosamine can be substituted for UDP-N-acetylgalactosamine, probably because of the presence of some UDP-N-acetylglucosamine epimerase. The amount incorporated in this instance is approximately 25% as great. If either UDP-N-acetylhexosamine or UDP-glucuronic acid is omitted, the incorporation of glucuronic acid or N-acetylgalactosamine, respectively, is diminished to 10% or less. When the reaction mixture is as described, the reaction proceeds in a linear fashion for about 20 minutes, with maximum incorporation of 0.5-1% at 1 hour. At 15 minutes' incubation time, the amount of radioactivity is proportional to the amount of microsomal preparation present over a range of 0.006-0.100 mg of protein per reaction mixture. The enzyme is active for at least two months when stored at --15°C. The microsomal preparation contains other enzymes which totally degrade the sugar nucleotide substratcs to unidentified products after 90 minutes of incubation. At 15 minutes, however, there is still a large proportion of undegraded sugar nucleotides in the reaction mixtures. II. Products Related to Heparin UDP-N-acetylglucosamine + UDP-glucuronicacid --* polysaccharide Assay Method
Principle. A microsomal preparation from heparin-synthesizing mouse mast cell tumors catalyzes the incorporation of N-acetyl-~H glucosamine and glucuronic acid-14C from UDP-N-acetyl-3H glucosamine and UDP-
494
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[84]
glucuronic acid-14C into a polysaccharide similar in sugar composition to hyaluronic acid, but resistant to degradation by hyaluronidase.5 The polysaccharide is possibly a precursor of heparin. Enzyme activity is assayed by isolation and counting of the radioactive polysaccharide.
Reagents UDP-N-acetyl-'~H glucosamine ( ~ 2 0 X 106 cpm/micromole) 5 UDP-glucuronic acid-14C (~10 >( 10~ cpm/micromole) 2 Tris, 0.5 M, pH 7.8 MgC12, 0.1 M Sucrose, 2 M Pancreatin, 1%, in 0.05 M Tris, pH 9.0
Procedure. ENZYMATICREACTION.The reaction mixture is prepared as follows in a volume made up to 0.04 ml: Tris (pH 7.8), 5 ~l; sucrose, 5 ~l; MgC12, 5 ~l; UDP-N-acetyl-StI glucosamine, 0.025 micromole (5 X 10~ cpm) ; UDP-glucuronic acid-14C, 0.025 micromole (2.5 X 10~ epm). The reaction is started by adding 0.01 ml of enzyme preparation containing about 0.3 mg of protein. After incubation at 37 ° for 2½ hours, the reaction is stopped by placing the tubes containing the reaction mixtures into a boiling water bath for 2 minutes. ISOLATION OF THE RADIOACTIVE POLYSACCtIARIDE. The isolation is identical to the isolation of radioactive chondroitin described in Section I. The radioactive material can be identified as a macromolecule by gel filtration2 It can be further identified and characterized by chromatography on DEAE-cellulose, by precipitation with cetyltrimethylammonium bromide, and by its resistance to degradation by testicular hyaluronidase.5 The radioactive polysaccharide is degraded by bacterial heparinase2 DETERMINATION Of THE RADIOACTIVITY. This determination is identical to the determination of radioactive chondroitin described in Section I. l~urification Procedure
Cultivation o] Mast Cell Tumors. Pieces of mouse mast cell tumor, 6 approximately 1 X 1 X 3 mm, are injected into DBA/2 mice by means of a 14-gauge needle with a trocar. The injections are best made subcutaneously along either or both flanks of the animal. Sterile conditions are not necessary. All the animals so inoculated will develop mast cell tumors. The best tumors are obtained 10-12 days after inoculation, when they have grown to good size (0.5-1.0 g each), but before they develop 5j. E. Silbert, J. Biol. Chem. 238, 3542 (1963). T. B. D u n n and M. Potter, d. Natl. Cancer Inst. 18, 587 (1957).
[84]
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCHARIDES
495
necrotic areas. The animals will die from the tumors on the 13th to 16th day after injection. Preparation of the Enzyme. The mice are killed by crushing the cervical spine with a clamp (hemostat). The skin of the flank is peeled back, and the adherent tumor is scraped from it with a scalpel. Tumor that is adherent to the rib cage is not taken. Tumors from 2 or 3 mice (2-4 g of tumor) are placed immediately in 25-50 ml of 0.25 M sucrose at 0-4 ° . All further steps are carried out at 0-4 ° . The tumors are minced with scissors until the pieces are approximately 2 mm 3 in size; they are then ground in a machine-driven Teflon Potter-Elvehjem homogenizer. The homogenate is centrifuged at 20,000 g for 10 minutes, and the supernatant is then centrifuged at 100,000 g for 20 minutes. After centrifugation, the supernatant is removed, the pellet is resuspended in 0.25M sucrose, and the preparation is centrifuged again at 100,000 g. The supernatant is removed, and the final pellet (usually about 0.3 ml) is suspended in an additional 0.25 ml of 0.25 M sucrose. Properties The incorporation of N-acetylglucosamine and glucuronie acid catalyzed by the microsomal preparation is equimolar and requires the presence of both UDP-N-acetylglucosamine and UDP-glucuronic acid. If either UDP-N-acetylglucosamine or UDP-glucuronic acid is omitted, the incorporation of glucuronic acid or N-acetylglucosamine, respectively, is diminished to 10% or less. UDP-glucosamine cannot be substituted for UDP-N-acetylglucosamine. When the reaction mixture is as described, the reaction proceeds in a linear fashion for about 30 minutes, with a maximum incorporation of 1-3% after 2 hours. At 20 minutes' incubation time, the amount of radioactivity is proportional to the amount of microsomal preparation present over a range of 0.10-1.50 mg of protein per reaction mixture. Incorporation of radioactivity is not stimulated by the presence of EDTA or mercaptoethanol; absence of Mg +÷ results in 50-75% less incorporation. The enzyme still retains activity after storage for one year at --15 ° . The microsomal preparation contains other enzymes which slowly degrade the sugar nucleotide substrates to unidentified products. After a 21/~ hour incubation only a small percentage of the substrates are left intact. At 30 minutes' incubation, most of the sugar nucleotides are still undegraded.
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCH&RIDES
491
o-amino acids a water-soluble product is formed. 1 These polymers spread on application to filter paper and are referred to as "spreading products." They yield only the monomer, GlcNAc-MurNAc(.L-ala.D-glu.mesoDAP.D-ala-D-ala) on hydrolysis with lysozyme. The presence of 1 or 2 D-alanine residues in such products is measured by the use of doubly labeled substrate containing 3H-meso-DAP and 14C-D-ala.14C-D-ala. The linear products synthesized in whole cells treated with penicillin or with D-amino acids have been seen by electron microscopy as linear strands/, s 7 C. Lark, D. Bradley, and K. G. Lark, Biochim. Biophys. Acta 78, 278 (1963). s p. Fitz-James and R. Hancock, J. Cell Biol. 26, 657 (1965).
[84] Animal Systems Synthesizing Mucopolysaccharides By JEREMIAH E. SILBERT I. Chondroitin UDP-N-acetylgalactosamine + UDP-glucuronic acid --* chondroitin Assay Method
Principle. A microsomal preparation from chick embryo cartilage catalyzes the incorporation of N-acetyl-3H galactosamine and glucuronic acid-14C from UDP-N-acetyl-~H galactosamine and UDP-glucuronic acid-14C into a polysaccharide having the characteristics of chondroitin? Enzyme activity is assayed by isolation and counting of the radioactive polysaccharide. Reagents UDP-N-acetyl-3H galactosamine ( ~ 2 0 X 106 cpm/micromole) ~ UDP-glucuronic acidJ4C ( ~ 1 0 X 106 cpm/micromole) 2 Tris, 0.5 M, pH 7.8 MgC12, 0.1 M Sucrose, 2 M Pancreatin, 1%, in 0.05 M Tris, pH 9.0
Procedure. ENZYMATIC REACTION.The reaction mixture is prepared as follows in a volume made up to 0.025 ml: Tris (pH 7.8), 5 t~l; MgCI~, 5 ~1; sucrose, 3 t~l; UDP-N-acetyl-~H galactosamine, 0.025 micromoles (5 X 105 epm) ; UDP-glucuronic acid-~4C, 0.025 micromoles (2.5 X 105 cpm). The reaction is started by the addition of 0.025 ml of enzyme prep1j. E. Silbert, J. Biol. Chem. 239, 1310 (1964). J. E. Silbert, Biochem. Biophys. Res. Commun. 9, 266 (1962).
492
ENZYMES OF C O M P L E X SACCHARIDE SYNTHESIS
[84]
aration containing about 0.05-0.07 mg of protein. After incubation at 37 ° for 90 minutes the reaction is stopped by placing the tubes containing the reaction mixtures into a boiling water bath for 2 minutes. ISOLATION OF THE RADIOACTIVE POLYSACCHARIDE. The total reaction mixture, including all the particulate material, is streaked in a 3-cm band on Whatman No. 1 chromatography paper. The ehromatograms are then developed overnight in a descending system, utilizing 95% ethanol-1 M ammonium acetate (5:2).3 All the radioactive sugar nucleotides and degradation products move varying distances down the paper, while the radioactive polysaccharide remains at the origin. The chromatogram is then dried, and the paper at the origin is cut out and incubated overnight at 37 ° in 5 ml of the pancreatin solution. This solubilizes protein-bound radioactive polysaccharide. An aliquot of the pancreatin digest is then dialyzed at room temperature for 24 hours against water with three changes of water. The volume of the liquid within the dialysis tubing is measured, and the liquid is then centrifuged at 20,000 g for 20 minutes. The clear supernatant contains the radioactive polysaccharide. The radioactive material can be identified as a macromolecule by gel filtration.1 I t can be identified as chondroitin by acid hydrolysis and identification of the sugar constituents, chromatography on DEAEcellulose, precipitation with cetyltrimethylammonium bromide, and susceptibility to degradation by various hyaluronidases.1 DETERMINATION OF THE RADIOACTIVITY.An aliquot of the clear supernatant is added to 15 ml of scintillation medium.4 Both 14C and 3H are determined simultaneously in a dual channel scintillation spectrometer. Alternatively, the dialysis step may be omitted, since all the radioactivity solubilized from the origin of the chromatogram remains within the dialysis tubing. However, the dialyzed preparation gives a somewhat more reliable assay because of quenching variability in counting of nondialyzed preparations. Purification Procedure Fertilized eggs are incubated at 37 ° in an incubator until the embryos are 13-15 days old. Each embryo is removed from its egg and its limbs are disarticulated from the pelvis. The muscle is stripped off, exposing both ends of each femur and tibia. The end cartilage at the joint surfaces is cut away with a fine scissors or scalpel blade, and the highly cellular epiphyses are expressed onto a glass plate by gentle ~A. C. Paladini and L. F. Leloir, Biochem. J. 51, 426 (1952). G. A. Bray, Anal. Biochem. I, 279 (1960).
[84]
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCHARIDES
493
pressure on the shaft of the bones 2-3 mm from the cut ends. The epiphyses are placed immediately in 5-10 ml of 0.25 M sucrose in an ice bath. Ordinarily, the time involved for processing 3{)-40 embryos has not exceeded 3 hours. The epiphyses are then homogenized in the sucrose solution at ()-4 ° with a Potter-Elvehjem glass homogenizer. The remainder of the enzyme preparation is carried out at {)-4° . The viscous homogenate is centrifuged at 10,000 g for l0 minutes, and the supernatant is then centrifuged at 100,000 g for 20 minutes. After centrifugation, the supernatant is removed, the pellet is resuspended in 0.25 M sucrose, and the preparation is centrifuged again at 100,000 g. The supernatant is removed and the final pellet (usually about 0.1 ml) is suspended in 0.25 ml of 0.25 M sucrose. Properties The incorporation of N-aeetylgalactosamine and glucuronic acid catalyzed by the microsomal preparation is equimolar, and the presence of both UDP-N-acetylgalactosamine and UDP-glucuronie acid is necessary for maximal formation. However, UDP-N-acetylglucosamine can be substituted for UDP-N-acetylgalactosamine, probably because of the presence of some UDP-N-acetylglucosamine epimerase. The amount incorporated in this instance is approximately 25% as great. If either UDP-N-acetylhexosamine or UDP-glucuronic acid is omitted, the incorporation of glucuronic acid or N-acetylgalactosamine, respectively, is diminished to 10% or less. When the reaction mixture is as described, the reaction proceeds in a linear fashion for about 20 minutes, with maximum incorporation of 0.5-1% at 1 hour. At 15 minutes' incubation time, the amount of radioactivity is proportional to the amount of microsomal preparation present over a range of 0.006-0.100 mg of protein per reaction mixture. The enzyme is active for at least two months when stored at --15°C. The microsomal preparation contains other enzymes which totally degrade the sugar nucleotide substratcs to unidentified products after 90 minutes of incubation. At 15 minutes, however, there is still a large proportion of undegraded sugar nucleotides in the reaction mixtures. II. Products Related to Heparin UDP-N-acetylglucosamine + UDP-glucuronicacid --* polysaccharide Assay Method
Principle. A microsomal preparation from heparin-synthesizing mouse mast cell tumors catalyzes the incorporation of N-acetyl-~H glucosamine and glucuronic acid-14C from UDP-N-acetyl-3H glucosamine and UDP-
494
ENZYMES OF COMPLEX SACCHARIDE SYNTHESIS
[84]
glucuronic acid-14C into a polysaccharide similar in sugar composition to hyaluronic acid, but resistant to degradation by hyaluronidase.5 The polysaccharide is possibly a precursor of heparin. Enzyme activity is assayed by isolation and counting of the radioactive polysaccharide.
Reagents UDP-N-acetyl-'~H glucosamine ( ~ 2 0 X 106 cpm/micromole) 5 UDP-glucuronic acid-14C (~10 >( 10~ cpm/micromole) 2 Tris, 0.5 M, pH 7.8 MgC12, 0.1 M Sucrose, 2 M Pancreatin, 1%, in 0.05 M Tris, pH 9.0
Procedure. ENZYMATICREACTION.The reaction mixture is prepared as follows in a volume made up to 0.04 ml: Tris (pH 7.8), 5 ~l; sucrose, 5 ~l; MgC12, 5 ~l; UDP-N-acetyl-StI glucosamine, 0.025 micromole (5 X 10~ cpm) ; UDP-glucuronic acid-14C, 0.025 micromole (2.5 X 10~ epm). The reaction is started by adding 0.01 ml of enzyme preparation containing about 0.3 mg of protein. After incubation at 37 ° for 2½ hours, the reaction is stopped by placing the tubes containing the reaction mixtures into a boiling water bath for 2 minutes. ISOLATION OF THE RADIOACTIVE POLYSACCtIARIDE. The isolation is identical to the isolation of radioactive chondroitin described in Section I. The radioactive material can be identified as a macromolecule by gel filtration2 It can be further identified and characterized by chromatography on DEAE-cellulose, by precipitation with cetyltrimethylammonium bromide, and by its resistance to degradation by testicular hyaluronidase.5 The radioactive polysaccharide is degraded by bacterial heparinase2 DETERMINATION Of THE RADIOACTIVITY. This determination is identical to the determination of radioactive chondroitin described in Section I. l~urification Procedure
Cultivation o] Mast Cell Tumors. Pieces of mouse mast cell tumor, 6 approximately 1 X 1 X 3 mm, are injected into DBA/2 mice by means of a 14-gauge needle with a trocar. The injections are best made subcutaneously along either or both flanks of the animal. Sterile conditions are not necessary. All the animals so inoculated will develop mast cell tumors. The best tumors are obtained 10-12 days after inoculation, when they have grown to good size (0.5-1.0 g each), but before they develop 5j. E. Silbert, J. Biol. Chem. 238, 3542 (1963). T. B. D u n n and M. Potter, d. Natl. Cancer Inst. 18, 587 (1957).
[84]
ANIMAL SYSTEMS SYNTHESIZING MUCOPOLYSACCHARIDES
495
necrotic areas. The animals will die from the tumors on the 13th to 16th day after injection. Preparation of the Enzyme. The mice are killed by crushing the cervical spine with a clamp (hemostat). The skin of the flank is peeled back, and the adherent tumor is scraped from it with a scalpel. Tumor that is adherent to the rib cage is not taken. Tumors from 2 or 3 mice (2-4 g of tumor) are placed immediately in 25-50 ml of 0.25 M sucrose at 0-4 ° . All further steps are carried out at 0-4 ° . The tumors are minced with scissors until the pieces are approximately 2 mm 3 in size; they are then ground in a machine-driven Teflon Potter-Elvehjem homogenizer. The homogenate is centrifuged at 20,000 g for 10 minutes, and the supernatant is then centrifuged at 100,000 g for 20 minutes. After centrifugation, the supernatant is removed, the pellet is resuspended in 0.25M sucrose, and the preparation is centrifuged again at 100,000 g. The supernatant is removed, and the final pellet (usually about 0.3 ml) is suspended in an additional 0.25 ml of 0.25 M sucrose. Properties The incorporation of N-acetylglucosamine and glucuronie acid catalyzed by the microsomal preparation is equimolar and requires the presence of both UDP-N-acetylglucosamine and UDP-glucuronic acid. If either UDP-N-acetylglucosamine or UDP-glucuronic acid is omitted, the incorporation of glucuronic acid or N-acetylglucosamine, respectively, is diminished to 10% or less. UDP-glucosamine cannot be substituted for UDP-N-acetylglucosamine. When the reaction mixture is as described, the reaction proceeds in a linear fashion for about 30 minutes, with a maximum incorporation of 1-3% after 2 hours. At 20 minutes' incubation time, the amount of radioactivity is proportional to the amount of microsomal preparation present over a range of 0.10-1.50 mg of protein per reaction mixture. Incorporation of radioactivity is not stimulated by the presence of EDTA or mercaptoethanol; absence of Mg +÷ results in 50-75% less incorporation. The enzyme still retains activity after storage for one year at --15 ° . The microsomal preparation contains other enzymes which slowly degrade the sugar nucleotide substrates to unidentified products. After a 21/~ hour incubation only a small percentage of the substrates are left intact. At 30 minutes' incubation, most of the sugar nucleotides are still undegraded.