Specific spectrophotometric assays for cathepsin B1

ANALYTICAL BIOCHEMISTRY 68, 119-127 (1975)

Specific Spectrophotometric Assays for Cathepsin BI1 A N D R E W S. BAJKOWSKI AND A L L E N F R A N K F A T E R

Department of Biochemistry and Biophysics, Loyola University Stritch School of Medicine, Maywood, Illinois 60153 Received January 13, 1975; accepted April 9, 1975 Cathepsin B 1 from bovine spleen was partially purified by acetone fractionation and by chromatography on Sephadex G-150 and D E A E Sephadex A-50. The enzyme was shown to catalyze the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl c~-N-benzyloxycarbonyl-L-lysinate. Under the assay conditions, cathepsin B1 is the major enzyme present in bovine spleen homogenates hydrolyzing these substrates. The kinetic parameters for the hydrolysis of p-nitrophenyl benzyloxycarbonylglycinate and p-nitrophenyl c~-Nbenzyloxycarbonyl-L-lysinate were measured and compared with those obtained for other cathepsin B 1 substrates. These results form the basis of an improved spectrophotometric assay for this enzyme in which the liberation of p-nitrophenol from either the N-benzyloxycarbonyl glycine or lysine p-nitrophenyl ester is monitored continuously at 326 nm.

Cathepsin B1 is a sulfhydryl protease found in the lysosomes of mammalian cells. It is thought to play a role in protein degradation in vivo (1) and may participate in other normal and pathological processes. Cathepsin B1 has been shown to degrade proteoglycans from adult human articular cartilage (2), native collagen (3), and can inactivate or otherwise modify several of the gluconeogenic and glycolytic enzymes from liver (4,5). Lysosomal proteases are thought to be mediators of inflammation (6,7) and may also play a role in cancer (8). Cathepsin B1 has been detected on the basis of its ability to hydrolyze certain esters and amides of N-substituted basic amino acids. Common synthetic substrates include BAA, 2 BAEE, B A N A , BAPA, and BLA (4). Protease activity has also been determined with denatured hemoglobin (9), edestin (10), gelatin (11), azo-casein (12), trypsinogen (13), and the oxidized/3-chain of insulin (14). 1 Supported in part by N I H General Research Grant RRO-5368. 2 BAA, c~-N-benzoyl-L-arginine amide; BAEE, a-N-benzoyl-L-arginine ethyl ester; BANA, c~-N-benzoyl-D,e-arginine 2-naphthylamide; BAPA, c~-N-benzoyl-D,L-arginine pnitroanilide; BLA, c~-N-benzoyl-L-lysine amide; C G N , c~-N-benzyloxycarbonyl glycine pnitrophenyl ester; CLN, t~-N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester; and E D T A [Ethylene dinitrilo] -tetraacetate. 119 Copyrightt~) 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.

124

BAJKOWSKI

AND

FRANKFATER

2

? '_o ~e I

I

~$ x 10 -5 M "L

FIG. 3. Lineweaver and Burk plot of rate data for the hydrolysis of CLN at 25°C. The buffer was 0.025 M sodium acetate, pH 5.1, containing 1.1 x 10 3 M E D T A +2.2 × 10-3 M dithiothreitol. r e s p e c t i v e l y (4). T h e r e w e r e at l e a s t t h r e e c o m p o n e n t s w i t h B A E E est e r a s e a c t i v i t y . O n e c o m p o n e n t p r o d u c e d a p e a k at t u b e 80 a n d m o s t l i k e l y c o r r e s p o n d s to c a t h e p s i n B1. A s e c o n d c o m p o n e n t p r o d u c e d a m i n o r p e a k at t u b e 60, a n d a t h i r d c o m p o n e n t p r o d u c e d a m a j o r p e a k at t u b e 23 c o r r e s p o n d i n g to t h e v o i d v o l u m e o f t h e c o l u m n . T h i s h i g h

16

12

o_ x

8

4

I

0.5

I

1.0 ~/S x I0 "5 M "1

I

1,5

FIG. 4. Lineweaver and Burk plot of rate data for the hydrolysis of CGN at 25°C. The buffer was 0.025 M sodium acetate, pH 5.1, containing 4.7 x 10-4 M E D T A +9.0 x 10-4 M dithiothreitol.

125

C A T H E P S I N B 1 ASSAYS

molecular weight sulfhydryl dependent BAEE esterase activity was observed in all our preparations and has not yet been characterized. Assays with BAPA, BANA, CLN, and C G N in each case revealed only a single major peak of activity under the conditions of the assay. The position of the peak in each case corresponded to the location of cathepsin B1. Chromatography on D E A E Sephadex A-50. Tubes 72-92 from the Sephadex G-150 column were pooled, applied to a D E A E Sephadex A-50 column, and eluted as described in the methods section. Fractions were assayed for protein at 280 nm and for BAPA, CLN, and C G N activity. The resulting elution profiles are shown in Fig. 2. As can be seen, within experimental error, the three activity profiles are coincident indicating that they are due to the same enzyme, cathepsin B , Determination of kinetic constants. Cathepsin Ba activity eluted from the D E A E Sephadex A-50 column was used throughout. Figure 3 represents a typical Lineweaver-Burk plot for the hydrolysis of CLN by cathepsin Ba. A similar plot for the hydrolysis of C G N is shown in Fig. 4. It is evident that the hydrolysis of CLN and C G N by cathepsin Ba obeys Michaelis-Menten Kinetics. Similar results were also seen with BAPA and BANA. The kinetic constants obtained with these various substrates are collected in Table I. The Km values reported here for the hydrolysis of BAPA and BANA by cathepsin B1 are in good agreement with the values obtained by other workers (4,20). From Table I it is also apparent that BANA is hydrolyzed 11 times faster than BAPA. A comparable ratio of reactivities of cathepsin B 1 toward these two amides has been seen by others (4,20). The kinetic parameters observed for the hydrolysis of C G N and CEN are also shown in Table I. It is apparent from their Km values that C G N and CLN bind 100 to 1000 times more strongly to cathepsin B1 than B A N A and BAPA. Similarly, a comparison of V values reveals that TABLE 1 KINETIC CONSTANTS FOR SOME CATHEPSIN B I SUBSTRATES AT p H 5.1 Va

Km

V/Km a

Substrate

(M sec -1 × 108)

(M X 106)

(sec × 105)

CLN b CGN c BANA a BAPA a

2080 + 203 337 -+ 33 61.2 5.38

2.7---.30 30.1 - 5.2 1560 1300

771,000 -+ 43,900 11,200 ± 1840 39.2 4.14

a Data corrected for differences' in E0. b Average of four determinations. c Average of three determinations. a Represents only a single determination.

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BAJKOWSKI AND FRANKFATER

C G N and C L N are hydrolyzed 5 and 30 times more rapidly than B A N A and 60 and 400 times more rapidly than BAPA. The fact that activated esters are hydrolyzed more rapidly than amides and with Km values several orders of magnitude smaller has been interpreted for other esterases and peptidases in terms of the formation of an acyl enzyme intermediate (21). The ratio V/Km which has been used as a measure of the specificity of an enzyme for a substrate is also presented in Table I. The values of V/Km for C G N and C L N are 290 and 20,000 times greater than the corresponding values for B A N A and 2700 and 190,000 times greater than the same values for BAPA.

DISCUSSION A number of assays have been previously devised for cathepsin B1 based on the ability of this enzyme to hydrolyze amide and ester derivatives of N-substituted arginine and lysine. In this paper we have shown that there are at least three components present in bovine spleen which hydrolyze BAEE. BAA is similarly hydrolyzed by at least two enzymes in tissue extracts, cathepsins B1 and B2 (4). BAPA appears to be relatively more specific for cathepsin Bi. However, the substrate is not hydrolyzed rapidly. Detection of small amounts of enzyme may therefore be difficult, requiring prolonged incubation times at elevated temperatures. In contrast, assays using B A N A are more sensitive. The release of fl-naphthylamine may be monitored spectrophotofluorometrically (20) or colorimetrically by coupling it to a diazonium salt (22). It has been suggested, however, that there may be a second enzyme present in tissue extracts with a molecular weight similar to cathepsin B1 which may also hydrolyze B A N A (23). In this paper we describe the use of C L N and C G N to assay for cathepsin Ba. C L N is a particularly favorable substrate being hydrolyzed 30 times faster than B A N A and 400 times faster than BAPA. In addition, owing to the poor Km values and sparing solubilities of BA N A and BAPA, under actual assay conditions C L N may be hydrolyzed 60 times more rapidly than B A N A and 800 times more rapidly than BAPA. Because of their favorable K,, values, cathepsin B1 may be assayed with C L N and C G N under conditions where So ~>Kin. Enzymatic rates obtained in this way are relatively insensitive to variations in substrate concentration and are linear with time for the duration of the major part of the reaction. REFERENCES 1. Tappel, A. L. (1969) in kysosomes in Biology and Pathology (Dingle, J. T., and Fell, H. B., eds.), Vol. 2, p. 207, North-Holland, Amsterdam. 2. Morrison, R. I., Barrett, A. J., Dingle, J. T., and Prior, D. (1973) Biochim. Biophys. Acta 302, 411.

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3. Burleigh, M. C., Barrett, A. J., and Lazarus, G. S. (1974) Biochem. J. 137, 387. 4. Otto, K. (1971) in Tissue Proteinases (Barrett, A. J. and Dingle, J. T., eds.), p. 1, North-Holland, Amsterdam. 5. Nakashima, K., and Ogino, K. (1974) J. Biochem. (Tokyo) 75, 355. 6. Lewis, G. P. (1964) Ann. N. Y. Acad. Sci. 116, 847. 7. Uvnas, B. (1964) Ann. N. Y. Acad. Sci. 116, 880. 8. Allison, A. C. (1969) in Lysosomes in Biology and Pathology (Dingle, J. T., and Fell, H. B., eds.), Vol. 2, p. 178, North-Holland, Amsterdam. 9. Anson, M. L. (1939) J. Gen. Physiol. 22, 79. 10. Snellman, O. (1969) Biochem. J. 114, 673. 11. Lundblad, G., and Falksrieden, L. G. (1964)Acta Chem. Scand. 18, 2044. 12. Bohley, P., Kirschke, H., Langner, J., Ansorge, H., Wiederanders, B., and Hanson, H. (1971) in Tissue Proteinases (Barrett, A. J. and Dingle, J. T., eds.), p. 1, NorthHolland, Amsterdam. 13. Greenbaum, L. M., Hirshkowitz, A., and Shoichet, I. (1959) J. Biol. Chem. 234, 2885. 14. Keilova, H. (1971) in Tissue Proteinases (Barrett, A. J. and Dingle, J. T., eds.), p. 29, North-Holland, Amsterdam. 15. Barrett, A. J. (1973) Biochem. J. 131, 809. 16. Farrell, T. J., Bajkowski, A. S., and Frankfater, A. (manuscript in preparation). 17. Seligson, D., and Seligson, H. (1951) J. Lab. Clin. Med. 38, 324. 18. Moore, S., and Stein, W. H. (1954) J. Biol. Chem. 211, 907. 19. Lineweaver, H., and Burk, D. (1934) J. Amer. Chem. Soc. 56, 658. 20. De Lumen, B. O., and Tappel, A. L. (1972) Anal. Biochem. 48, 378. 21. Zerner, B., and Bender, M. L. (1964) J, Amer. Chem. Soc. 86, 3669. 22. Barrett, A. J. (1972) Anal. Biochem. 47, 280. 23. De Lumen, B. O., and Tappet, A. L. (1972) J. Biol. Chem..247, 3552.