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[56] β-Aspartyl peptidase from rat liver
[56]
~-ASPARTYL PEPTIDASE FROM RAT LIVER
737
mum, but the rat enzyme is more active in phosphate than in Tris buffers. Enzyme activity was not detected in human serum.
[56] ]~-Aspartyl Peptidase from Rat L i v e r 1 B y EDWARD E. HJa~EY
Assay Method Principle. The glyeine formed in the enzymatic reaction was estimated by a photometric ninhydrin methodY Assay Method B for fl-aspartyl peptidase from Escherichia coli was adapted from this assay method with slight modifications. Reagents
Sodium phosphate buffer, a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~. The pH depends on the dilution. fl-Aspartylglycine, 0.02 M Dowex 50W-X4, 100-200 mesh, hydrogen form Dowex 2-X8, 100-200 mesh, acetate form Pyridine, 2 M Citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide--methyl Cellosolve reagent 3 Ethyl alcohol, 60% Procedure. The reaction mixtures had a total volume of 2.0 ml and consisted of the enzyme sample, 100 micromoles of sodium phosphate buffer, and 5 micromoles of fl-aspartylglycine. The peptide was omitted from the blank. The final pH was 7.2. Incubation was at 37 ° for 2 hours and the reaction was stopped by heating in a boiling water bath for 5 minutes. After centrifugation 1.5 ml of the supernatant was added to a 1 X 2.5 cm column of Dowex 50W-X4. The column was washed with 8.5 ml of water and eluted with 10 ml of pyridine solution. The pyridine eluate was evaporated to dryness under vacuum and the residue dissolved in 5 ml of water. This solution was added to a 1 X 2.5 cm column of Dowex 2-X8, and the column was washed with 15 ml of water. The effluent was evaporated to dryness, and the residue analyzed for glycine by the ninhydrin method described in Method A for the E. coli enzyme.
F. E. Dorer, E. E. Haley, and D. L. Buchanan, Arch. Biochem. Biophys. 127, 490 (1968). *A. T. Matheson and B. L. Tattrie, Can. J. Biochem. 42, 95 (1964); E. W. Yemm and E. C. Cocking,Analyst 80, 209 (1955). s Prepared as described in the chapter on fl-aspartyl peptidase from E. coli.
738
PEFrIDASES
[56]
Definition o] Unit of Enzyme Activity. One unit of enzyme activity is defined as that amount required to hydrolyze 1 micromole of fl-aspartylglycine in 1 minute at 37 ° under the conditions of the assay. Purification Procedure All operations except CM-cellulose and hydroxylapatite chromatography and the heat treatment were carried out in the cold. Step 1. Extraction. Albino male rats, 300-400 g, which h'ad been maintained on Purina chow pellets, were killed by decapitation, and the livers quickly removed. About 50 g of liver was blended with 3 volumes of cold 0.1 M sodium phosphate buffer (a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~) for 30 seconds in a prechilled stainless steel Waring blendor. The homogenate was centrifuged to remove cell debris? The supernatant (fraction A, Table I) was centrifuged for 1 hour at 105,000 g in a Spinco Model L ultracentrifuge. Step 2. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to the supernatant (fraction B, 18 mg of protein/ml) to 40% saturation (0.243 g/ml). The mixture was stirred for another 30 minutes and the suspension was centrifuged. More ammonium sulfate was added to the supernatant to 55% saturation (0.097 K/ml). The mixture was stirred an additional 30 minutes and the suspension was centrifuged. The precipitate was dissolved in 20 ml of 0.1 M sodium phosphate buffer and dialyzed against 5 liters of this buffer for 16 hours. Step 3. Heat Treatment. The dialyzed solution (fraction C) was immersed in a water bath at 60 ° for 5 minutes, cooled, centrifuged, and the supernatant dialyzed against 5 liters of 0.005 M sodium phosphate buffer for 16 hours. The turbid solution was centrifuged. Step 4. CM-Cellulose Chromatography. The supernatant from above (fraction D) was applied to a 2.3 X 23 cm column of Whatman CM 32 equilibrated with 0.005 M sodium phosphate buffer. The column was eluted with the same buffer. The fractions from a wide yellow band were combined and adjusted to 0.01 M sodium phosphate buffer concentration. Step 5. Hydroxylapatite Chromatography. The above eluate solution (fraction E) was applied to a 2.3 X 11 cm column of Bio-Gel HTP 5 equilibrated with 0.01 M sodium phosphate buffer. The column was eluted with 0.05 M sodium phosphate buffer and the first 120 ml of efltuent discarded. The fractions from a narrow yellow band, which was eluted with 1 M potassium phosphate buffer, pH 7.5, were combined and dialyzed against 5 li.ters of 0.01 M sodium phosphate buffer for 16 hours (fraction F). ' Centrifugation was at 600 g for 1 hour in the cold unless otherwise stated. Purchased from Bio-Rad Laboratories, New York, New York.
[$5]
~-ASPARTYL PEPTIDAS~. FROM RAT LIVER
789
TABLE I PARTIAL PURII~ICATION OF ~-AsP.CRTYL PEFrIDASE FROM RAT LIVER"
Specific Fraction A. "Low-speed" supernatant B. 105,000 g Supernatant C. Ammonium sulfate (40-55% satn) D. Heat at 60° E. CM-cellulose F. Hydroxylapatite
Protein ~ (rag)
Activity (units)
activity (units/mg)
Yield (%)
Purification (-fold)
3300
1.55
0.00047
(100)
1.0
2370 570
0.55 0.70
0.00065 0. 0012
100 45
1.4 2.6
216 147 72
0.60 0.55 0.50
0.0028 0. 0037 0. 0070
39 35 32
6.0 8.0 14.9
, Starting with 50 g (wet weight) of liver. Protein was determined by the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A summary of the purification data is given in Table I. Properties
Stability. The enzyme activity is stable for as long as 5 days at room temperature, and is stable to freezing and thawing in 0.01 M sodium phosphate buffer. Stability is dependent upon the presence of sodium ions. The activity is unstable to dialysis against water or 0.01 M Tris-HC1 buffer, pH 8.0. Activity cannot be restored by the addition of NaC1, but if NaC1, 0.01 M, is included in the dialysis solution, 807~ of the activity is recovered. Purity. The enzyme preparation, fraction F, represents a 15-fold purification over the liver homogenate. The extent of purification with regard to separation from a-aspartylglyeine and leucylglycine hydrolyzing activities, however, was 500- and 40-fold, respectively. Attempts to purify the fl-aspartyl peptidase activity further by DEAE-cellulose chromatography were unsuccessful due to loss of activity on the column. Specificity. The hydrolysis of peptides other than fl-aspartyl peptides appears to be due to contaminating enzymes present in the preparation, because of the relative enrichment toward fl-aspartylglycine activity in the course of the purification. In Table II is shown the action of the enzyme on various types of substrates, and the relative difference in activities between fractions B and F toward fl-aspartyl peptides and other peptides. The fl-aspartyl tripeptides, fl-aspartylglycylglycine, -glycylalanine, and -glycylvaline, were cleaved at the aspartyl bond. Activators and Inhibitors. The peptidase appears to require sodium or potassium for maximal activity, but not alkaline earth or transition
740
PEPTIDASES
[55]
TABLE II HYDROLYSIS OF PEPTIDES BY RAT LIVER PREPARATIONS Relative hydrolysis~ (% of ~-aspartylglycine)
Peptide ~-Aspartylglycine ~-Aspartyla]anine ~-Aspartylvaline ~-Aspartylleucine ~-Aspartylisoleucine ~-Aspar tylserine ~-Aspartylthreonine ~-Aspartylmethionine 8-Aspartylglycylglycine~ fl-Aspartylglyeylalanine ~ fl-Aspar tylglycylvaline a-Aspartylglycine a-Aspar tylalanine a-Aspar tylleucine a-Aspartylserine a-Aspartylglycylglycine a-Aspar tylglycylalanine v-Glutamylleucine a-Glutamylleuc~ne Asparagine Glycylglycine Glycylalanine Glycylvaline Glycylleucine Leucylglycine Glycylglycylglycine ~-Alanylglycine N-Acetylglycine Carnosine
Fraction Bb
Fraction F~
Method of analysis
(100)
(100) 51 2865 37 56 29 82 95 55 13 3 10 9 10 3 3 0 5
• e e e e e e e e e e e e e e • e e e
0
f
59 47
1500
2000 1000
40 4O Trace Trace 50 30 0 0 0
g g g g g g g g g
a All incubations were carried out as described for ~-aspartylglycine under assay method and included a substrate blank for each peptide to correct for any free amino acid present in or formed from substrate in the absence of enzyme. b Enzyme fractions are described in Table I. c At the end of the incubation of ~-aspartylglycylglycine, glycylglycine was identified by paper chromatography IF. E. Doter, E. E. Haley, and D. L. Buchanan, A n a l Biochem. 19, 366 (1967)] in a desalted aliquot of the incubation mixture. Glycylalanine from ~oaspartylglycylalanine was identified as above. • The extent of hydrolysis was determined by ninhydrin reaction of the Dowex 2 effluent as described for ~-aspartylglycine under assay method. The results were corrected for differences in the ninhydrin color values of the various substrate C-terminal amino acids or dipeptides.
[57]
PEPTIDE HYDROLASES
741
metal ions. There was a slight activation by mercaptoethanol, while inhibition was caused by p-hydroxymercuribenzoate but not by iodoacetamide. pH Optimum and Buf]er Ef]ects. The pH optimum is 7.5-8.0 in sodium phosphate buffer. There is a broad pH optimum between 7 and 9 in Tris-HC1 buffer, and the activity is about half of that in the sodium phosphate buffer. Distribution. Homogenates of rat kidney, brain, lung, skeletal muscle, and heart muscle, in addition to liver, all catalyze a slow but significant hydrolysis of fl-aspartylglycine. Rat kidney enzyme, prepared under the same conditions as for fraction B of liver, showed a specificity similar to that of the liver enzyme. The specific activity of the kidney enzyme is about twice that of the liver enzyme at the same stage of purification? * See also the discussion of distribution of fl-aspartyl peptidase in Chapter [55] on the E. coli enzyme.
1 Ammoniawas determined by Nesslerization of an aliquot of the incubation mixture. The extent of hydrolysis was estimated by semiquantitative determination of hydrolysis products by paper chromatography in a desalted aliquot of the incubation mixture.
[57] Peptide Hydrolases in Mammalian Connective Tissue By CHRISTIAN SCHWABE Connective tissue constitutes about 20% of the body pro~ins of a macroorganism. Its function is as varied at its appearance in topographically different areas such as skin, bone, or as the supporting tissue of various organs. The cellular element of the connective tissue, the fibroblasts, are responsible for the production and maintenance of the intercellular components, collagen, and acid mucopolysaccharides. Peculiarly, in spite of its substantial contribution to the total body weight, large portions of homogeneous cellular connective tissue are rare. A notable exception is the pulp of teeth which consists of fibroblasts, collagen, and mucopolysaccharides with a negligible amount of endothelium (capillaries) and nerve fibers interwoven. This tissue provides an unambiguous source of fibroblasts needed for the study of connective tissue peptide hydrolases. The peptide bond-hydrolyzing enzymes found in connective tissue are divided into two groups, the neutral peptide hydrolases and the acid proteases, according to the hydrogen ion concentration at which they are
~-ASPARTYL PEPTIDASE FROM RAT LIVER
737
mum, but the rat enzyme is more active in phosphate than in Tris buffers. Enzyme activity was not detected in human serum.
[56] ]~-Aspartyl Peptidase from Rat L i v e r 1 B y EDWARD E. HJa~EY
Assay Method Principle. The glyeine formed in the enzymatic reaction was estimated by a photometric ninhydrin methodY Assay Method B for fl-aspartyl peptidase from Escherichia coli was adapted from this assay method with slight modifications. Reagents
Sodium phosphate buffer, a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~. The pH depends on the dilution. fl-Aspartylglycine, 0.02 M Dowex 50W-X4, 100-200 mesh, hydrogen form Dowex 2-X8, 100-200 mesh, acetate form Pyridine, 2 M Citrate buffer, 0.067 M, pH 5.0 Ninhydrin-potassium cyanide--methyl Cellosolve reagent 3 Ethyl alcohol, 60% Procedure. The reaction mixtures had a total volume of 2.0 ml and consisted of the enzyme sample, 100 micromoles of sodium phosphate buffer, and 5 micromoles of fl-aspartylglycine. The peptide was omitted from the blank. The final pH was 7.2. Incubation was at 37 ° for 2 hours and the reaction was stopped by heating in a boiling water bath for 5 minutes. After centrifugation 1.5 ml of the supernatant was added to a 1 X 2.5 cm column of Dowex 50W-X4. The column was washed with 8.5 ml of water and eluted with 10 ml of pyridine solution. The pyridine eluate was evaporated to dryness under vacuum and the residue dissolved in 5 ml of water. This solution was added to a 1 X 2.5 cm column of Dowex 2-X8, and the column was washed with 15 ml of water. The effluent was evaporated to dryness, and the residue analyzed for glycine by the ninhydrin method described in Method A for the E. coli enzyme.
F. E. Dorer, E. E. Haley, and D. L. Buchanan, Arch. Biochem. Biophys. 127, 490 (1968). *A. T. Matheson and B. L. Tattrie, Can. J. Biochem. 42, 95 (1964); E. W. Yemm and E. C. Cocking,Analyst 80, 209 (1955). s Prepared as described in the chapter on fl-aspartyl peptidase from E. coli.
738
PEFrIDASES
[56]
Definition o] Unit of Enzyme Activity. One unit of enzyme activity is defined as that amount required to hydrolyze 1 micromole of fl-aspartylglycine in 1 minute at 37 ° under the conditions of the assay. Purification Procedure All operations except CM-cellulose and hydroxylapatite chromatography and the heat treatment were carried out in the cold. Step 1. Extraction. Albino male rats, 300-400 g, which h'ad been maintained on Purina chow pellets, were killed by decapitation, and the livers quickly removed. About 50 g of liver was blended with 3 volumes of cold 0.1 M sodium phosphate buffer (a molar mixture of 3 parts Na2HP04 and 1 part NaH2P0~) for 30 seconds in a prechilled stainless steel Waring blendor. The homogenate was centrifuged to remove cell debris? The supernatant (fraction A, Table I) was centrifuged for 1 hour at 105,000 g in a Spinco Model L ultracentrifuge. Step 2. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to the supernatant (fraction B, 18 mg of protein/ml) to 40% saturation (0.243 g/ml). The mixture was stirred for another 30 minutes and the suspension was centrifuged. More ammonium sulfate was added to the supernatant to 55% saturation (0.097 K/ml). The mixture was stirred an additional 30 minutes and the suspension was centrifuged. The precipitate was dissolved in 20 ml of 0.1 M sodium phosphate buffer and dialyzed against 5 liters of this buffer for 16 hours. Step 3. Heat Treatment. The dialyzed solution (fraction C) was immersed in a water bath at 60 ° for 5 minutes, cooled, centrifuged, and the supernatant dialyzed against 5 liters of 0.005 M sodium phosphate buffer for 16 hours. The turbid solution was centrifuged. Step 4. CM-Cellulose Chromatography. The supernatant from above (fraction D) was applied to a 2.3 X 23 cm column of Whatman CM 32 equilibrated with 0.005 M sodium phosphate buffer. The column was eluted with the same buffer. The fractions from a wide yellow band were combined and adjusted to 0.01 M sodium phosphate buffer concentration. Step 5. Hydroxylapatite Chromatography. The above eluate solution (fraction E) was applied to a 2.3 X 11 cm column of Bio-Gel HTP 5 equilibrated with 0.01 M sodium phosphate buffer. The column was eluted with 0.05 M sodium phosphate buffer and the first 120 ml of efltuent discarded. The fractions from a narrow yellow band, which was eluted with 1 M potassium phosphate buffer, pH 7.5, were combined and dialyzed against 5 li.ters of 0.01 M sodium phosphate buffer for 16 hours (fraction F). ' Centrifugation was at 600 g for 1 hour in the cold unless otherwise stated. Purchased from Bio-Rad Laboratories, New York, New York.
[$5]
~-ASPARTYL PEPTIDAS~. FROM RAT LIVER
789
TABLE I PARTIAL PURII~ICATION OF ~-AsP.CRTYL PEFrIDASE FROM RAT LIVER"
Specific Fraction A. "Low-speed" supernatant B. 105,000 g Supernatant C. Ammonium sulfate (40-55% satn) D. Heat at 60° E. CM-cellulose F. Hydroxylapatite
Protein ~ (rag)
Activity (units)
activity (units/mg)
Yield (%)
Purification (-fold)
3300
1.55
0.00047
(100)
1.0
2370 570
0.55 0.70
0.00065 0. 0012
100 45
1.4 2.6
216 147 72
0.60 0.55 0.50
0.0028 0. 0037 0. 0070
39 35 32
6.0 8.0 14.9
, Starting with 50 g (wet weight) of liver. Protein was determined by the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). A summary of the purification data is given in Table I. Properties
Stability. The enzyme activity is stable for as long as 5 days at room temperature, and is stable to freezing and thawing in 0.01 M sodium phosphate buffer. Stability is dependent upon the presence of sodium ions. The activity is unstable to dialysis against water or 0.01 M Tris-HC1 buffer, pH 8.0. Activity cannot be restored by the addition of NaC1, but if NaC1, 0.01 M, is included in the dialysis solution, 807~ of the activity is recovered. Purity. The enzyme preparation, fraction F, represents a 15-fold purification over the liver homogenate. The extent of purification with regard to separation from a-aspartylglyeine and leucylglycine hydrolyzing activities, however, was 500- and 40-fold, respectively. Attempts to purify the fl-aspartyl peptidase activity further by DEAE-cellulose chromatography were unsuccessful due to loss of activity on the column. Specificity. The hydrolysis of peptides other than fl-aspartyl peptides appears to be due to contaminating enzymes present in the preparation, because of the relative enrichment toward fl-aspartylglycine activity in the course of the purification. In Table II is shown the action of the enzyme on various types of substrates, and the relative difference in activities between fractions B and F toward fl-aspartyl peptides and other peptides. The fl-aspartyl tripeptides, fl-aspartylglycylglycine, -glycylalanine, and -glycylvaline, were cleaved at the aspartyl bond. Activators and Inhibitors. The peptidase appears to require sodium or potassium for maximal activity, but not alkaline earth or transition
740
PEPTIDASES
[55]
TABLE II HYDROLYSIS OF PEPTIDES BY RAT LIVER PREPARATIONS Relative hydrolysis~ (% of ~-aspartylglycine)
Peptide ~-Aspartylglycine ~-Aspartyla]anine ~-Aspartylvaline ~-Aspartylleucine ~-Aspartylisoleucine ~-Aspar tylserine ~-Aspartylthreonine ~-Aspartylmethionine 8-Aspartylglycylglycine~ fl-Aspartylglyeylalanine ~ fl-Aspar tylglycylvaline a-Aspartylglycine a-Aspar tylalanine a-Aspar tylleucine a-Aspartylserine a-Aspartylglycylglycine a-Aspar tylglycylalanine v-Glutamylleucine a-Glutamylleuc~ne Asparagine Glycylglycine Glycylalanine Glycylvaline Glycylleucine Leucylglycine Glycylglycylglycine ~-Alanylglycine N-Acetylglycine Carnosine
Fraction Bb
Fraction F~
Method of analysis
(100)
(100) 51 2865 37 56 29 82 95 55 13 3 10 9 10 3 3 0 5
• e e e e e e e e e e e e e e • e e e
0
f
59 47
1500
2000 1000
40 4O Trace Trace 50 30 0 0 0
g g g g g g g g g
a All incubations were carried out as described for ~-aspartylglycine under assay method and included a substrate blank for each peptide to correct for any free amino acid present in or formed from substrate in the absence of enzyme. b Enzyme fractions are described in Table I. c At the end of the incubation of ~-aspartylglycylglycine, glycylglycine was identified by paper chromatography IF. E. Doter, E. E. Haley, and D. L. Buchanan, A n a l Biochem. 19, 366 (1967)] in a desalted aliquot of the incubation mixture. Glycylalanine from ~oaspartylglycylalanine was identified as above. • The extent of hydrolysis was determined by ninhydrin reaction of the Dowex 2 effluent as described for ~-aspartylglycine under assay method. The results were corrected for differences in the ninhydrin color values of the various substrate C-terminal amino acids or dipeptides.
[57]
PEPTIDE HYDROLASES
741
metal ions. There was a slight activation by mercaptoethanol, while inhibition was caused by p-hydroxymercuribenzoate but not by iodoacetamide. pH Optimum and Buf]er Ef]ects. The pH optimum is 7.5-8.0 in sodium phosphate buffer. There is a broad pH optimum between 7 and 9 in Tris-HC1 buffer, and the activity is about half of that in the sodium phosphate buffer. Distribution. Homogenates of rat kidney, brain, lung, skeletal muscle, and heart muscle, in addition to liver, all catalyze a slow but significant hydrolysis of fl-aspartylglycine. Rat kidney enzyme, prepared under the same conditions as for fraction B of liver, showed a specificity similar to that of the liver enzyme. The specific activity of the kidney enzyme is about twice that of the liver enzyme at the same stage of purification? * See also the discussion of distribution of fl-aspartyl peptidase in Chapter [55] on the E. coli enzyme.
1 Ammoniawas determined by Nesslerization of an aliquot of the incubation mixture. The extent of hydrolysis was estimated by semiquantitative determination of hydrolysis products by paper chromatography in a desalted aliquot of the incubation mixture.
[57] Peptide Hydrolases in Mammalian Connective Tissue By CHRISTIAN SCHWABE Connective tissue constitutes about 20% of the body pro~ins of a macroorganism. Its function is as varied at its appearance in topographically different areas such as skin, bone, or as the supporting tissue of various organs. The cellular element of the connective tissue, the fibroblasts, are responsible for the production and maintenance of the intercellular components, collagen, and acid mucopolysaccharides. Peculiarly, in spite of its substantial contribution to the total body weight, large portions of homogeneous cellular connective tissue are rare. A notable exception is the pulp of teeth which consists of fibroblasts, collagen, and mucopolysaccharides with a negligible amount of endothelium (capillaries) and nerve fibers interwoven. This tissue provides an unambiguous source of fibroblasts needed for the study of connective tissue peptide hydrolases. The peptide bond-hydrolyzing enzymes found in connective tissue are divided into two groups, the neutral peptide hydrolases and the acid proteases, according to the hydrogen ion concentration at which they are