Calf Tendon Procollagen Peptidase: Its Purification and Endopeptidase Mode of Action (Dermatosparaxis/Antiprocollagen Peptidase/Antiprocollagen) LEONARD D

Home , Collagenase, Procollagen peptidase

Proc. Nat. Acad. Sci. USA Vol. 71, No. 1, pp. 40-44, January 1974

Calf Tendon Procollagen Peptidase: Its Purification and Endopeptidase Mode of Action (dermatosparaxis/antiprocollagen peptidase/antiprocollagen) LEONARD D. KOHN*, CHAVIVA ISERSKY*, JAMES ZUPNIKt, ALBERT LENAERSt, GEORGE LEE*t, AND CHARLES M. LAPIEREt *National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and tService de Dermatologie, Hopital de Baviere, University of Liege, 4000 Liege, Belgium Communicated by Leon A. Heppel, September 4, 1973

ABSTRACT The procollagen peptidase activity of calf MATERIALS AND METHODS tendon has been purified. The enzyme has a high degree of specificity for native procollagen and converts both Collagen Substrates and Enzyme Assay. Dermatosparaxic pro al and pro a2 to al and a2, respectively. The purified procollagen served as the substrate in all reactions; its prepa- enzyme is an endopeptidase which excises the amino ration has been described (5, 13). The standard enzyme assay terminal peptide extensions of the precursor chains in ml solution (0.6 mg of native block; the molecular size and amino-acid composition of included 0.1 of the substrate the excised peptides compare favorably with those pre- procollagen per ml), 0.05 ml of M glycyl glycine buffer (pH dicted in previous reports. Antisera to the enzyme and to 7.5), and appropriate aliquots of enzyme, in a final volume procollagen have been prepared and have been used to of 0.25 ml. Tubes were incubated at 26° for 6 hr unless other- characterize the enzyme, the enzymatically excised pep- wise noted. Reactions were stopped and analyzed by disc-gel tides, and the enzyme-peptide complex in reaction mix- tures. electrophoresis as described (13). All assays were run at multiple protein concentrations and in duplicate. Dermatosparaxis is an inherited connective tissue disorder of Enzyme activity was quantitated by calculating the frac- cattle which is characterized by extreme fragility of the skin tion of pro al or pro a2 converted to al or a2 when compared (1, 2). The dermis of these animals contains disorganized to control incubations without enzyme. Since each incubation collagen bundles which demonstrate an impaired parallel mixture contained 60 jig of intact native procollagen, the packing of individual collagen filaments within the collagen fractional conversion was readily expressed in absolute terms, fibers (3, 4). These abnormal fibers are presumed to result i.e., one unit of enzyme activity was defined as the conversion of from the accumulation of procollagen, the triple helical 1 Mig of native procollagen per 6 hr per assay volume. Specific precursor of collagen which is composed of pro al and pro activity correlated these units to enzyme protein measured a2 chains (5-9). Pro al and pro a2 chains are ribosomal calorimetrically (15). which contain an additional peptide of 100 products (10, 11) Immunization and Evaluation of Antisera. New Zealand residues at the amino termini of their respective a to 200 rabbits were immunized with native procollagen using sched- chains (5, 8, 9, 12). described elsewhere (16). Antisera were In a previous report (13) we described an enzyme activity ules and techniques which excised the amino-terminal regularly evaluated by microimmunodiffusion (16, 17) or in normal bovine tissues immunoelectrophoresis (16,18,19). extension of the precursor chains to yield electrophoretically used in this chains. We further showed that this activity was The unadsorbed, antiserum to procollagen normal report reacted with native procollagen but gave no reaction absent or markedly reduced in dermatosparaxic tissues, i.e., and the resultant tissue with collagen or normal calf serum in immunodiffusion or by the accumulation of procollagen radioimmunoassay (16). changes were apparently caused by a defect in a specific enzyme, "procollagen peptidase." The "procollagen pep- RESULTS tidase" activity was associated with a very low level of non- Purification of the enzyme protease activity in contrast to pepsin and chymo- specific of killed calves were trypsin, two enzymes used in vitro by several laboratories Tendons obtained from the legs freshly al carefully cleaned of contaminating muscle and were washed to convert pro al to chains (6, 7, 14). M NaCl. In we describe the of calf- three times with 0.05 M Tris HCl (pH 7.4)-0.15 the present report purification 0-2' as tendon procollagen peptidase, characterize several of its The dissecting and washing procedure was done at properties, and show that the enzyme is an endopeptidase were all subsequent manipulations. Tendon preparations were the amino-terminal of in either processed immediately or stored at -70° for subsequent which excises peptide procollagen in results. block rather than in multiple fragments. extractions with no significant difference Step 1: Homogenization. Tendon pieces were homogenized in a Hobart commercial meat To cool I This work was performed in partial fulfillment of the degree by grinding grinder. requirements for the Department of Chemistry, American the tissue during this procedure, and to aid in its processing, University, Washington, D.C. 20016. frozen pieces of the wash buffer were alternated with tissue 40 Proc. Nat. Acad. Sci. USA 71 (1974) Tendon Procollagen Peptidase 41

aliquots. The total amount of frozen buffer used was two to three times the weight of the tendon homogenized.

was Step 2: Centrifugation of Homogenate. The homogenate 2. centrifuged at 7500 X g for 15 min. The supernatant solution was pooled and could be stored at -20° for as long as 3 months with no significant change in enzyme activity.

Step 3: Ammonium Sulfate Precipitation. Ammonium sul- oioo--~ o-o-o\ J fate, 39 g/100 ml, was slowly added to the thawed supernatant 10- St.2 ~2 3 4 5 6 1200 of Step 2. The stirred suspension was equilibrated overnight 1.0 100 and the precipitate was harvested by centrifugation at co 40,000 X g for 20 min. The precipitate was solubilized in < I.A°*,-*st 11000 0.05 M Tris HCl (pH 7.4)-i mM mercaptoethanol-0.15 M NaCl-10 mM CaC12. CC 0I8 800 Step 4: Dialysis. By dialysis for 24 hr against two separate 0.6 '_ 0- 600 6-liter volumes, the solubilized precipitate was equilibrated with the buffer used in Step 3 minus NaCl. The flocculant - precipitate which formed under these conditions was removed 0.4- B C D 20H400

by centrifugation at 40,000. X g for 10 min. 0.2 -l10 200 Step 5: DEAE-Cellulose Chromatography. The supernatant from was a X 4-cm solution Step 4 applied (1 ml/min) to 100 -~~~~- column of microgranular DEAE previously equilibrated with 0 100 200 FRACTION 0.05 M Tris HCl (pH 7.4),-1 mM mercaptoethanol. The FIG. 1. (A) DEAE-Cellulose chromatography of procollagen was a column eluted with linear gradient consisting of 0-0.3 peptidase. Fractions of 20 ml were collected at a flow rate of 1-2 M NaCl in equilibrating buffer (Fig. 1A). ml/min. Procollagen peptidase activity (@--*) was eluted as a peak between fractions 220 and 280. Arrow indicates the ini- Step 6: Ammonium Sulfate Extraction. Enzyme activity tiation of the NaCl gradient. The ordinate on the right is units eluted from the DEAE column (Fig. IA, black bar) was pooled, of enzyme activity. (B) Sephadex G-200 chromatography of concentrated by ultrafiltration, and precipitated by the addi- DEAE-cellulose purified enzyme. Fractions of 12 ml were col- tion of ammonium sulfate to a final concentration of 60%. lected at a flow rate of 0.5 ml/min. Disc-gel inserts are: (1) the The precipitate was harvested by centrifugation at 40,000 X 10% ammonium sulfate extract (Step 6) of the DEAE-cellulose g and was consecutively extracted by the following solutions: enzyme pool, 50 yg; (2-4) peaks A, B, and C, respectively, of 40, 30, 25, 20, and 10% ammonium sulfate in equilibrating the Sephadex G-200 chromatography procedure, 50Mg; (5) peak buffer (Step 5) and equilibrating buffer alone. The supernatant A of the Sephadex G-200 procedure after incubation with 1% of the extraction with the highest specific activity (usually sodium dodecylsulfate, 50 Mg; and (6) peak A of the Sephadex G-200 procedure after incubation with 1% sodium dodecylsulfate the 10% extract) was dialyzed against 0.05 M Trisp Cl, (pH plus 0.1 M mercaptoethanol, 50Oug. The first four gels use a pH 7.5)-i mM mercaptoethanol-10 mM CaCl2-0.15 M NaCl. 8.5 system (20); the last two use a sodium dodecylsulfate gel system described by Maizel (21). The anode is at the bottom Step 7: Sephadex G-200 Chromatography. The enzyme con- of the gel in all cases; the stain used was Coomassie blue (22). centrate from Step 6 was applied to a 200 X 2.5-cm column of Sephadex G-200 previously equilibrated with the dialysis A summary of the purification procedure is presented in buffer given in Step 6. Elution was at 20-30 ml/hr (Fig. 1B). Table 1. The addition of phenylmethylsulfonyl fluoride (0.1 TABLE 1. Purification of calf-tendon procollagen peptidase

Total Total Specific Specific Volume protein activity* % activity activity Step (ml) (i-ng) (U) Recovered (U/mg) increase 1. Crude 3,400 1,145,000 480,000 0.4 1 2. Crude supernate 3,000 16,5)00 450,000 94 27.3 68 3. Ammonium SO4 350 5,800 440,000 92 75.9 190 4. Dialysis 380 2,195 340,000 71 154.9 387 5. DEAE 820 280 169,000 35 603.6 1,509 6. Ammonium SO4 10% extract 52 130 104,000 22 800 2,000 7. Sephadex G-200 Pk A 5 68 52,760} 775.9 1,939 Pk B 5 35 26,446 21 755.6 1,889 Pk C 5 25 20,000 800 2,000

* Independent measurements of both the pro al and the pro a2 conversion gave similar results; therefore, the average of these results is presented. U = units of enzyme activity; 1 unit is defined as the conversion of 1 ,ug of native procollagen per 6 hr per assay volume. 42 Cell Biology: Kohn et al. Proc. Nat. Acad. Sci. USA 71 (1974)

A Peptide Procollogen

F _liii A I pro aI-: a pro a20 2 i

...... --

i. B +e a ...;

FIG. 2. (A) Disc-gel analyses of an aliquot of dermato- i2 sparactic procollagen before (left) and after (right) incubation with the peak A enzyme. The 6-hr digestion resulted in complete con-

version of the pro a l to al and pro a2 to a2. Aliquots of the same Enzyme Bound Peptide mixture or its duplicate have been used for the experiments in FIG. 3. (A and B) Immunoelectrophoresis of heat denatured Figs. 2B, 3 4. at top and The anode is the of the gels. (B) procollagen (1) and enzyme treated procollagen (2) against Immunodiffusion assay of procollagen, 3 mg/ml (1); enzyme antiserum to procollagen (troughs). A is unstained; B is the treated 1.5 sham procollagen, mg/ml (2); incubated procollagen, same as A but stained with amido Schwartz. Note that in Pattern 1.5 mg/ml (3); and enzyme (4) against to antiserum procollagen 2 of both A and B there is a significant amount of enzyme cleaved, (center well). immunoreactive peptide which coelectrophoresed with enzyme (enzyme-bound peptide). If the enzyme treated procollagen is mg/ml) to all steps of the procedure did not significantly alter exposed to 2 M urea before electrophoresis, the immunoreactive the results. peptide is released from the enzyme and migrates with "free" peptide. Characterization of the enzyme Gel filtration on Sephadex G-200 resolved the enzyme prepara- component as well as the appearance of a 140,000 10,000 tion into four distinguishable protein peaks, three of which molecular weight component. had enzyme activity (Fig. 1B). Each active peak had a similar Antisera were produced to the peak A enzyme preparation protein pattern when analyzed by disc-gel electrophoresis from Sephadex G-200 and were absorbed with lyophilized (20), i.e., two major components, one of which had an RF of preparations of normal calf sera. The absorbed antisera pre- 0.33 and one of which just barely penetrated (RF < 0.05) the cipitated the enzyme (Table 2) but the enzyme-antibody running gels (Fig. 1B). Both components were enzymatically precipitate retained its procollagen-peptidase activity. Im- active when eluted from the gels (750-850 units/mg). munoelectrophoresis indicated that the enzyme-antisera Electrophoresis of an aliquot from any G-200 peak (A, B, interaction resulted in an elongated, humped precipitin line, and C; Fig. 1B) on gels containing sodium dodecylsulfate i.e., the procollagen peptidase preparations contained at least (21) indicated that each peak contained a component with two molecular species with common antigenic determinants. molecular weight 70,000 i 5,000 and a component which There was no antisera reaction with calf sera or procollagen. barely penetrated 5% gels. Sodium dodecylsulfate electro- The resolution of the enzyme activity into three fractions phoresis of the same samples after they were preincubated of similar specific activity by molecular-sieve chromatography, (21) with sodium dodecylsulfate and 0.1 AI mercaptoethanol their similar disc-gel patterns, the sodium dodecylsulfate gel showed an increase in the 70,000 5000 molecular weight data, and the immunoelectrol)horetic data all suggested that TABLE 2. Neutralization of procollagen peptidase activity by the adsorbed antiserutn* to procollagen peptidase (PCPase)

Starting Normal PCPase activity PCPase activity Total PCPase PCPase rabbit recovered in recovered in activity activity Antiserum sera the supernatant the precipitate recovered Tube (total units) (ml) (ml) (total units) (total units) (total units) 1 160 0.1 - 40 111 151 2 160 0.05 - 110 29 139 3 160 0.025 138 14 152 4 160 0.1 150 0 150 5 160 0.05 148 0 148 6 160 0.025,=) 155 0 155

Each tube contained 0.2 ml of the Step 7 enzyme (Fig. 1B, peak A) and 0.1 ml of buffer containing varying amounts of antisera or normal rabbit sera. The buffer was 0.05 M Tris - HCl (pH 7.4)-0.15 iAT NaCi. Tubes were incubated for 1 hr at, room temperature and for 16 hr at 2-4° before the supernatant solution and precipitate were separated by centrifugation at 20,000 X g. Both the supernatant solution and the precipitate resuspended in the above buffer were assayed for procollagen peptidase activity. * Antisera were adsorbed by adding 25 mg of a dialyzed and lyophilized 60% ammonium sulfate precipitate of normal calf serum, by incubating for 18 hr at 40, and by centrifugation for 1 hr at 12,000 X g to remove the precipitate. Proc. Nat. Acad. Sci. USA 71 (1974) Tendon Procollagen Peptida'se 43 TABLE 3. Amino-acid composition of the peptide excised from dermatosparaxic procollagen by procollagen peptidase (residues/peptide)

Pro al Peptide Pro a2 As excised* Residualt Peptide As by after excised* by procollagen collagenase procollagen Amino acid peptidase digestion peptidase Hydroxylysine 0 0 0 Lysine 4.7 6 3.2 Histidine 1.6 2 1. 4 Arginine 3.3 4 1. 5 4-Hydroxyproline 0 0 1. 2 Aspartic 25. 3 27 10.6 Threonine 9.4 10 3.8 Serine 2.9 3 4.8 Glutamic 33.8 37 9. 8 Proline 19 22 8.8 Glycine 15 16 19.3 Alanine 3.9 3 8.6 Haff-cystine 14.31 16 4.3 Valine 15.1 17 4.1 I,00o 1 -. Methionine 0 0 0 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Isoleucine 4.7 4 1.9 RF Leucine 6.2 5 2.9 FIG. 4. Gel electrophoresis in sodium dodecylsulfate (25) of Tyrosine 1.6 2 0.4 the disc-gel eluates containing the immunoreactive peptides Phenylalanine 1.4 1 1.8 formed by the action of procollagen peptidase (0) or heat denaturation (A). Standards (d) were: bovine-serum albumin (1); * Data are based on 18-, 36-, and 72-hr hydrolyzate data. ovalbumin (2); carbonic anhydrase (3); trypsin (4); hemoglobin Hydrolysis samples contained 100 lug and were prepared as (5); cytochrome c (6); pancreatic-trypsin inhibitor (7); and described (26). A Beckman model 120C analyzer was used. glucagon (8). The anode is at the bottom; staining was with Coomassie blue. Gels were 12.5% in acrylamide. t As deternmined by Furthmayr et al. (12). t Based on samples oxidized with performic acid (27). diffusion studies against antisera to procollagen (Fig. 2B). Since the antisera to procollagen were specific for the amino- the purified calf-tendon procollagen peptidase preparations terminal peptide extension (16, 24),. these immunoreactive contained polymeric protein components with molecular peptides apperared to have been excised as intact moieties. weights of 70,000 and 140,000 which were catalytically active. Confirmation of this presumption was provided by im- Kinetic Properties. The purified enzyme (peaks A, B, or C; munoelectrophoretic data. Immunoelectrophoresis of native Fig. 1B) was maximally active between pH 7.0 and 7.5 and procollagen demonstrated the presence of one major precipitin catalyzed the conversion of both precursor chains, pro al and band (16), whereas immunoelectrophoresis of heat denatured pro a2, to their respective a chains (Fig. 2A). Km values for procollagen demonstrated the presence of two major precipitin pro al and pro a2 in dermatosparaxic procollagen molecules bands; one remains near the application well as does native approximated 10-6 M; Km values for pro al and pro a2 as procollagen, and one migrates to the anode (Fig. 3A and B, individual chains approximated 10-1 M, i.e., the enzyme Pattern 1). The anodal band consisted of two peptides with showed a significant preference for precursor chains in a molecular weights 11,500 i 3000 and 17,500 + 2500 on triple helical array rather than random coiled precursor sodium dodecylsulfate gels. chains isolated by CMI-cellulose chromatography (5, 23). In contrast, immunoelectrophoresis of native procollagen, Enzyme activity was partially inhibited by mercapto- treated with enzyme, demonstrated the presence of two major ethanol (01 M), glutathione (0.01 M), p-hydroxymercuri- anodal precipitin bands and the marked loss of the native benzoate (2.5 mM), Mn2+ (0.1 M), and Mg2+ (0.1 M). Ca2+ procollagen band around the application well (Fig. 3A and B, between 0.1 M and 0.01 mM and 1% Triton X-100 had no Pattern 2). The most anodal band was identical in location to effects. Diisopropylfluorophosphate at a 1000-fold molar the anodal band of heat denatured procollagen, i.e., repre- excess did not inhibit enzyme activity. Enzyme concentra- sented the immunoreactive peptides of procollagen which had tions which completely converted pro al and pro a2 to al and been excised in block and released from the enzyme (see below). a2, respectively, (Fig. 2A), had no significant peptidase The less anodal band migrated in the area of procollagen activity against casein, albumin, collagen, and myoglobin. peptidase and contained immunoreactive peptides which had been excised by the enzyme but which had remained bound Endopeptidase action of the enzyme to it. This was readily demonstrated by exposure of the reac- Despite complete enzymatic conversion of pro al to al and pro tion mixture to 2 M urea which released the bound peptides, a2 to a2 (Fig. 2A), there was no significant loss in immuno- i.e., eliminated the less anodal band and moved its immuno- reactivity when procollagen treated with enzyme was com- reactive material into the most anodal band containing the pared to native or heat denatured procollagen in immuno- excised immunoreactive peptides of procollagen (not shown). 44 Cell Biology: Kohn et al. Proc. Nat. Acad. Sci. USA 71 (1974)

The immunoreactive peptides released by the action of the ence or absence of the collagen moiety; quantitative radioim- calf-tendon procollagen peptidase were isolated from disc gels munoassay data support this suggestion (16). run at pH 8.5. On these gels the peptides ran just above the 1. Hanset, R. & Ansay, M. (1967) Ann. Med. Vet. 7,451-459. dye front and could be eluted from unstained gels. Immuno- 2. Hanset, R. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352, logic analyses of eluates from sliced gels showed that slices 13. from the upper and lower portions of the immunoreactive 3. O'Hara, P. J., Read, W. K., Romane, W. M. & Bridges, area on these gels contained individual peptides, whereas C. H. (1970) Lab. Invest. 23, 307-314. slices from the middle of the contained 4. Simar, L. J. & Betz, E. H. (1971) Hoppe-Seyler's Z. Physiol. immunoreactive area Chem. 352, 13. both peptides. Aliquots of the eluates containing the indivi- 5. Lenaers, A., Ansay, M., Nusgens, B. V. & Lapiere, C. M. dual peptides were subjected to amino-acid analysis (Table 3) (1972) Eur. J. Biochem. 23, 533-543. and to sodium dodecylsulfate gel analysis (Fig. 4). The results 6. Bellamy, G. & Bornstein, P. (1971) Proc. Nat. Acad. Sci. for the enzymatically excised pro al and pro a2 amino- USA 68, 1138-1142. 7. Layman, D. L., McGoodwin, E. B. & Martin, G. R. (1971) terminal peptides compare favorably with the amino-acid Proc. Nat. Acad. Sci. USA 68,454-458. compositions and molecular weights previously predicted 8. Dehm, P., Jimeney, S. A., Olsen, B. R. & Prokop, D. J. for in block excisions. (1972) Proc. Nat. Acad. Sci. USA 69,60-64. 9. Bornstein, P., von der Mark, K., Wyke, A. W., Ehrlich, DISCUSSION H. P. & Monson, J. M. (1972) J. Biol. Chem. 247, 2808- 2813. The present report described the purification of calf-tendon 10. Kerwar, S. S., Kohn, L. D., Lapiere, C. M. & Weissbach, procollagen peptidase and characterizes several of its proper- H. (1972) Proc. Nat. Acad. Sci. USA 69, 2727-2731. ties. The purified enzyme is not homogeneous by disc-gel 11. Lazarides, E. & Lukens, L. N. (1971) Nature New Biol. analyses; however, evidence is presented to suggest that the 232, 37-40. 12. Furthmayr, H., Timpl, R., Stark, M., Lapiere, C. M. & nonhomogeneity is related to the existence of polymeric Kuhn, K. (1972) FEBS Lett. 28, 247-250. enzyme forms. The presumption of our past experiments (13) 13. Lapiere, C. M., Lenaers, A. & Kohn, L. D. (1971) Proc. has been that the enzyme acts as an endopeptidase and Nat. Acad. Sci. USA 68,3054-3058. cleaves the pro al and pro a2 chains in block. In the present 14. Bornstein, P., Ehrlich, H. P. & Wyke, A. W. (1972) Science 175,544-546. report we show that the purified enzyme yields peptides 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, which are immunologically intact and have the molecular R. J. (1951)J. Biol. Chem. 193, 265-275. weight and amino-acid composition predicted for in block 16. Isersky, C., Lee, G., Lapibre, C. M. & Kohn, L. D., J. excisions of the pro al and pro a2 amino-terminal peptides. Immunol., manuscript submitted. The peptides released by heat denaturation of procollagen 17. Crowle, A. J. (1961) Immunodiffusion (Academic Press, New York). (Fig. 3A and B, Pattern 1) can quantitatively account for 18. Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. Immunol. the al and a2 chains present in dermatosparaxic extracts. 7, 103-110. The al and a2 chains present in the dermatosparaxic pro- 19. Grabar, P. & Burtin, P. (1960) Analyse Immunodlectro- collagen preparations do not, therefore, indicate contamina- phoretique (Masson et Cie, Editeurs, Paris). 20. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427. tion by al and a2 but rather the existence of cleaved precursor 21. Maizel, J. V. (1966) Science 151,988-990. chains which have not "released" their peptides. Their re- 22. Chrambach, A., Reisfield, R. A., Wyckoff, M. & Zaccari, J. lease prior to heat denaturation is apparently restricted by (1967) Anal. Biochem. 20, 150-154. conformational restraints imposed by the native triple helical 23. Piez, K. A., Eigner, E. A. & Lewis, M. S. (1963) Biochemistry structure. 2,58-66. the ex- 24. Timpl, R., Wick, G., Furthmayr, H., Lapiere, C. M. & Immunodiffusion assays of intact procollagen and Kuhn, K. (1973) Eur. J. Biochem. 32, 584-591. cised peptide indicated that the amino-terminal peptide ex- 25. Neville, D. M. (1971) J. Biol. Chem. 246,6328-6334. tension of procollagen retained its immunogenic activity after 26. Kohn, L. D., Warren, W. A. & Carroll, W. R. (1970) J. complete enzyme digestion. This suggests that this peptide Biol. Chem. 245, 3821-3830. has a stable conformation which is independent of the pres- 27. Hirs, C. H. W. (1956) J. Biol. Chem. 219, 611421.