Cardioiwsculur Research, 1979. 13, 621 -629
Degradation of canine cardiac myosin and actin by cathepsin D isolated from homologous tissue EDWARD A . O G U N R O , R I C H A R D B. LANMAN, J U L I A R. S P E N C E R , ALAN G . FERGUSON, A N D MICHAEL LESCH From the Department of’ Medicine, Section of Cardiology, Northwestern University Medical School, Chicago, Illinois
The ability of canine cardiac cathepsin D to catalyse hydrolysis of myosin andactin isolated from homologous tissue was monitored using both SDS polyacrylamide gel electrophoresis and the release of trichloroacetic acid soluble radioactivity from the radioactively (14C) labelled proteins. Cathepsin D readily catalysed the hydrolysis of myosin and actin. The pH-activity profile for myosin hydrolysis exhibited two optima with a major peak at pH 2.6 together with a smaller peak at pH 3.4. This was distinct from the pH-activity profile for actin hydrolysis where a single optimum at pH 4.2 was observed. Incubation of cathepsin D with myosin (pH 2.6, 37°C) resulted in complete degradation of myosin light chains (MW = 2 8 0 0 0 ~ 7 0 0and 18600k350, n = 4 ) within 10 min while heavy chains (MW= 190400f2500, n=4) were degraded into three proteins of molecular weight 32OOOi-750; 37000k1000 and 49000k750 (n-4) within 4 h. Further incubation (24 h) of cathepsin D with myosin resulted in two proteins of MW 29OOOk2000 and 48800k3100 (n=4). In the case of actin (MW 44800k 1600, n=6) polypeptides produced as a result of hydrolytic cleavage by cathepsin D (pH 4.2, 37°C) were not observed on SDS gels, although the overall size of the actin band was markedly decreased at 24 h. Cathepsin D catalysed the hydrolysis of myosin at near neutral pH (6.5) although the overall rate of degradation was considerably reduced compared to that observed at pH 2.6. Pepstatin A was a potent inhibitor of the hydrolysis of myosin and actin at all pH values investigated indicating that cathepsin D and not a contaminant was responsible for the observed degradation.
SUMMARY
Cardiac lysosomal acid proteases are thought to play an essential role in cell death following ischaemic injury (Ricciutti, 1972; Welman and Peters, 1977; Wildenthal, 1978) and in the turnover of myocardial proteins (Morgan et al., 1974). At the present time however, the specific role of these enzymes in the above degradative processes is incompletely defined. This arises as a consequence of a number of reasons. Firstly, little data is available on the susceptability of major myocardial proteins to hydrolysis by lysosomal acid proteases.Secondly, many acid proteases exhibit pH optima that are considerably lower than the pH values thought to exist within the lysosome (Reijngoud and Tager, 1973; Ohkuma and Poole, 1978). These observations together with reports of the presence of myocardial tissue proteases with neutral (Reddy et al., 1975) and alkaline (Griffin and Wildenthal, 1978) pH optima, have focused conReprint requests to: Edward A. Ogunro, Ph.D., Department
of Medicine, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, Illinois 6061 I , USA.
siderable attention upon the need to define the role of lysosomal acid proteases in mammalian myocardial protein catabolism. The aim of the studies reported in this paper were twofold: 1) To study whether cathepsin D (EC 3.4.4.23, a major lysosomal acid protease) isolated from myocardial tissue catalyses the degradation of myofibrillar proteins such as myosin and actin isolated from homologous tissue. 2) To investigate whether the isolated cathepsin D is capable of catalysing the hydrolysis of myosin or actin at pH values close to physiological pH. Materials and methods All reagents were of the highest analytical grade commercially available and were obtained from either Fisher Scientific, Itasca, Illinois; Scientific Products, McGaw Park, Illinois, or The Sigma Chemical Company, St. Louis, Missouri. Radioactively labelled chemicals were obtained from New England Nuclear, Boston, Massachusetts. 62 1
622
Edward A. Ogunro, Richuril B. Latinrun, Jiilia H. Spmwr, Alun ti. Fwgusorr, untl Micliud L i w l i
PURIFICATION OF CATHEPSIN D Cathepsin D was purified approximately 2500 fold to homogeneity from canine cardiac tissue using techniques previously developed (Ogunro et a/., 1979) for the isolation of this enzyme from rabbit myocardium. Hearts were removed from dogs by performing a right thoracotomy following chloralose anaesthesia. The hearts were washed with sodium chloride (1 50 mmol.litre-l) to remove blood, trimmed, blotted dry, minced, and finally homogenised for eight periods of 15 s at 15 s intervals (Waring Blender, top speed, 4 C) in three volumes of a solution containing sodium chloride ( I 50 mmol. litre-]) and butanol (270 mmol*litre-I). Triton X-100 was added to the homogenate to give a final concentration of 0.1 % w/v (in order to maximise disruption of lysosomes) and the homogenate was stirred (30 min) and centrifuged at 7000 g for 20 min. The pellet (PI) was discarded and the supernatant (S1) was further centrifuged at IOOOOO g for 60 min. The pellet (obtained following 1OOOOO g centrifugation, P,) was discarded and the supernatant (S,)was dialysed for 12 h against acetate buffer (100 mmol. litre-], pH 3.7) containing sodium chloride (500 mrnol*litre-') in order to remove the butanol and Triton X-100. During dialysis some protein precipitation occurred and this was removed by centrifugation at IOOOOO g for 15 rnin. The supernatant (S,) from this latter centrifugation was filtered through a 10 pm filter (Millipore Corporation, Bedford, Massachusetts) and concentrated to approximately 10 cms. The concentrated solution (50 to 60 mg p r o t e i n a ~ - ~was ) run onto a pepstatinylsepharose affinity column prepared according to the procedure of Murakami and lnagami (1975) and was continuously washed with sodium acetate buffer (100 mmol*litre-l, pH 3.7 NaCl 500 mmol.litre-l until the absorbance (280 nm) of the column effluent was less than 0.05. The eluting buffer was then changed to bicarbonate (100 mmol.litre-l, pH 8.3 /NaCI 50 mmol.litre-l) and 10 fractions of 5 cms volume were collected. The fractions with highest specific activity of acid protease were pooled, concentrated to approximately 1 cms (6 to 12 mg protein*~m-~) and further purified by gel filtration chromatography (sephacryl S200) using the above bicarbonate buffer as eluant. Twenty fractions of 3 cms volume were collected. The fractions with the highest specific activity of acid protease were again pooled, concentrated to a volume of 2 cms (3 to 6 mg p r o t e i n a r a ) dialysed against deionised water for 12 h (two changes) and finally subjected to isoelectric focusing (Radola, 1973) using pH gradient of 3.5 to 10.0. During isoelectric focusing, major cathepsin D activity equilibrated between pH values of 7.0 and 7.6 and these fractions were pooled and
stored (pH 7.0 to 7.6) at -20 C. Under these conditions the enzyme was stable for periods up to 4 months. PURIFICATION OF MYOSIN
Myosin was purified from canine cardiac tissue according to the procedure of Shiverick et a/. ( 1975) with the exception that ATP (2.5 mmol.litre-l) was added to the myosin homogenisation - extraction solution to facilitate the dissociation of actomyosin and thereby increase the yield of myosin (Mommaerts, 1958). PURIFICATION OF G-ACTIN The procedure of Szent-Gyorgyi ( 1947) was used to prepare an acetone powder from canine myocardium. Subsequent steps in the purification procedure involved extraction of G-actin from the acetone powder, polymerisation to form F-actin, depolymerisation and gel filtration using sephadex G-200 (Rees and Young, 1967). RADIOACTIVE LABELLING OF MYOSIN AND ACTIN
Myofibrillar proteins were radioactively (I4C) labelled using the reductive alkylation procedure of Rice and Means (1971). Following labelling, protein solutions were dialysed to remove radioactive byproducts. Solutions of myosin were dialysed (40 h, 3 changes) against imidazole buffer ( 10 mmol-litre-l, pH 6.8) containing KCI (0.5 mol.litre-l) and dithiothreitol (2.0 mmol.litre-l) while solutions of actin were similarly dialysed against an ATP solution (0.5 mmol-litre-', pH 7.5) which contained 2mercaptoethanol (0.5 mmol*litre-l) and CaCI, (0.2 mmol*litre-I). In general, solutions of labelled myosin were freshly prepared for each experiment, however actin samples were stored (4 'C) for periods of up to 2 weeks. PROTEIN DETERMINATIONS
Two methods of protein determination were used. In studies with myosin, the ratio of absorbance at 280 nm and 260 nm was employed. In studies with actin the procedure of Lowry et a/. (1951) was used with crystalline human serum albumin as standard. EVALUATION OF MYOSIN A N D ACTIN AS SUBSTRATES FOR CATHEPSIN D
Two assay procedures were employed : 1) Quantitation of the release of trichloroacetic acid (TCA) soluble radioactivity from radioactively labelled myosin and actin. 2) Analysis of degradation products of myosin and actin using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. This method
623
Degradarion of myosin and actin hv cathepsin D
was employed in addition to the radioactive procedure since it offered the possibility of a greatly improved assay sensitivity in situations where large (TCA insoluble) polypeptides may have been produced following protease action. In the first method, radioactively labelled myofibrillar protein and cathepsin D were incubated (37'C, 30 min) at a ratio of 70:1 w/w respectively. The reaction mixture (0.2 cms) contained myosin (0.20to 0.25 mg) or actin (0.20to 0.25 mg), cathepsin D, sodium acetate buffer (0.2 mol-litre-', pH 2.0 to 7.0) and KCI (0.5mol*litre-l). Following incubation, the reaction was terminated by precipitation of all proteins with 0.2cm3TCA (0.6mol*litre-') and the assay mixture was further incubated (10 min) at 0°C to ensure complete precipitation. The samples were centrifuged (lOO000 g x 10 min) in a microcentrifuge (Beckman Instruments, Irvine, California) and the radioactivity in 0.1 cm3 aliquots of the supernatant fraction was determined (Packard Tricarb Scintillation Counter Model 3375,Packard Instrument Company, Inc., Downers Grove, Illinois) using 10 cms Instagel Scintillation mixture (Packard Instrument Company, Inc.). In the second method non radioactively labelled myofibrillar protein and cathepsin D were incubated (37"C,10 min to 24 h) as described above, however the ratio of myofibrillar protein to cathepsin D was increased to 4O:l w/w to improve sensitivity for detection of degradation products of myosin or actin. Following incubation the reaction was terminated by cooling the mixture to 0°C. Samples containing myosin were dialysed against a solution of imidazole (10 mmol.litre-l, pH 6.8) containing KCI (0.5 mol*Iitre-l), while samples of actin were dialysed against a solution of ATP (0.5mmol*litre-l, pH 7.5) containing 2-mercaptoethanol (0.5 mollitre-') and CaC12 (0.2 mmol.litre-'). SDS electrophoresis was subsequently performed using the method of Weber and Osborn (1969).In both assay procedures blank determinations were carried out in which distilled water was substituted for cathepsin D. Since there has been some indication of protease activity associated with actin preparations (Drabikowski, 1961),all actin samples were heated to 100°C for 10 min before use in order to inactivate such enzyme activity. Sodium acetate buffer was employed throughout the pH ranges investigated in order to minimise possible differential effects of various buffer solutions on the pH-activity profile for cathepsin D. N o detectable change in pH was observed during the incubation period at any of the pH values investigated. D ACTIVITY Cathepsin D was assayed as described by Anson ASSAY OF CATHEPSIN
(1938).
MOLECULAR WEIGHT DETERMINATIONS
Molecular weight determinations using the SDS electrophoretic technique of Weber and Osborn (1969)were performed on the purified preparations of myosin and actin and on the degradation products of these proteins following incubation with cathepsin D. Two groups of standards were employed : 1) High molecular weight standards: Myosin heavy chain (MW =200000), galactosidase ( 1 3oooO), phosphorylase B (940001, bovine serum albumin (68000) and ovalbumin (43000).These proteins were obtained as a kit from Bio-Rad, Laboratories Inc., Richmond, California. 2) Low molecular weight standards: Human serum albumin (68000), pepsinogen (43000),a chymotrypsinogen A (25500) and cytochrome C (12380).
Results EVALUATION OF MYOSIN A N D ACTIN PREPARATIONS
Myosin and actin were typically isolated from canine cardiac tissue with yields of 5 m g p and 1 mg.gl tissue wet weight respectively. The Anea:Aoso ratio of the isolated myosin was usually greater than 1.45 and the specific activity of the radioactively labelled proteins was of the order of 1 x loe dpmmg-I protein for myosin and 1 x lo5 d p m m g l protein for actin. On SDS polyacrylamide gels the purified myosin was represented by a single heavy chain of molecular weight 190400f2500, n=4, a number of minor proteins of molecular weight approximately 12oooO and two light chains of MW 28000 f700 and 18600 h350 n =4. SDS electrophoresis of purified actin revealed the presence of a single polypeptide of molecular weight 44800 f1600, n =6. The values calculated for the molecular weights of myosin heavy and light chains and actin were in good agreement with values previously published for the molecular weights of these proteins isolated from cardiac (Sarkar et a/., 1971 ; Uchida et a/.,1977)and other tissues (Rees and Young, 1967;Sender, 1971). EVALUATION OF CATHEPSIN PREPARATION
D
SDS electrophoretic studies of cathepsin D isolated from canine myocardial tissue revealed the presence of two polypeptides of molecular weight approximately 32000 and 14OOO daltons (Fig. 1). The smaller polypeptide accounted for approximately 20% of the total protein and probably represented a subunit of the enzyme since it was not observed following polyacrylamide gel electrophoresis performed in the absence of sodium dodecyl sulphate.
Edward A. Ogunro, Richard B. Lanman, Julia R. Spencer, Alan G . Ferguson. and Michael Lesch
624
myosin hydrolysis (Fig. 3a) was bimodal with a major optimum at pH 2.6 together with a smaller peak (approximately 80% of the first peak) at pH 3.4. These results were distinctly different from the pH-activity profile for the hydrolysis of actin (Fig. 3b) where a single optimum at a pH of approximately 4.2 was observed. 2) Studies with unlabelledproteins a ) Myosin The cathepsin D mediated degradation of
Fig. 1 Typical results obtainedfollowing SDS electrophoresis (under reducing conditions) of the purified canine cardiac cathepsin D. E V A L U A T I O N OF M Y O S I N A N D A C T I N AS SUBSTRATES FOR CATHEPSIN D
1) Studies with labelled proteins Cathepsin D readily catalysed the degradation of myosin and actin as verified by the progressive release of TCA soluble radioactive peptides from the radioactively labelled proteins (Fig. 2). pH optimum studies revealed that the pH-activity profile for
--
a) M Y O S W
b) ACTIN
w 60 -
Fig. 2 Linearity of a ) myosin and b) actin hydrolysis by cathepsin D using the radioactively labelled myofibrillar proteins as substrates. Results represent the mean of duplicate determinations. Studies were carried out by incubation of 0.20 to 0.25 mg of myosin or actin (2.9 to 3.6 pg cathepsin D) at p H values of 2.6 and 4.2 respectively. Control studies were carried out by substituting distilled water for the enzyme in 1 -. L - - 1 -_Idthe assay mixture followed by 60 20 40 60 80 100 niin (myosin) or 100 min (actin) incubation, INCUBATION TIME ( m m )
120 -
too
INCUBATION TIME (min )
myosin at pH 2.6 (as monitored using SDS electrophoresis) is shown in Fig. 4a. Myosin light chains were completely degraded within 10 min of incubation while heavy chains were degraded into proteins of molecular weight 32000f750; 37000 t lo00 and 490003~750daltons (n=4) within 4 h. Further incubation at this pH (up to 24 h) resulted in a negligible change in molecular weight of the 49000 dalton protein, however the 37000 and 32000 dalton proteins were further degraded and appeared as a diffuse protein band of MW 29000+2000 (n=4). The results of studies in which the hydrolytic activity of cathepsin D towards myosin was compared at pH values ranging from’2.6 to 6.5 are shown in Fig. 4b. Cathepsin D catalysed the degradation of myosin at all the pH values investigated, however the overall rate of hydrolysis was considerably reduced as the pH of the incubation medium was increased to 6.5. At pH 5.0 myosin was degraded within 24 h into three major proteins of molecular weight 33000 1500; 40000 3000 and 49000flSOO (n=4). It is evident from Fig. 6 that polypeptides of similar molecular weight are also produced following incubation at pH values of 5.4 and 5.9. At pH 6.5 however, the overall rate of degradation is considerably reduced such that the formation of these polypeptides can not be seen at 24h.
625
DeRradation of ni.vosin and actin by cathepsin D _ _ ~
._
a) MYOSIN
b) ACTIN
~
2o O2.0
t
6.0 01 20
3.0L 4.0 - 5.0 pH OF ASSAY
I
I
I
5.0 pH OF ASSAY
3.0
4.0
A A
B
C
D
L, 6.0
B
Fig. 3 pH-activity profiles for cathepsin D using radioactively labelled a) myosin and b) actin as substrates. Results represent the mean of duplicate determinations. Studies were carried out by incubation ofmyosin or actin and cathepsin D, as in legend to Fig. 2. at the appropriate p H values followed by 30 min incubation. Control studies were performed by substitution of distilled water for the enzyme at each p H value investigated followed by a similar periodof incubation.
C
D
E
F
E
Fig.4a Typical results obtained following incubation of cathepsin D with myosin at p H 2.6 and subsequent SDS electrophoresis (under reducing conditions). A) Control. B ) 10 min, C ) 30 min, D ) 4 h and E ) 24 h incubation. Studies were carried out by incubating 0.20 to 0.25 nig of myosin with 5.0 to 6.5 pg cathepsin D for the indicated time period. Control studies were performed 6.v substitution of distilled water for the enzyme followed by 24 h incubation.
Fig. 4b Typical results obtained following incubation (24 h) of cathepsin D and myosin at various p H values. A ) Control, B ) p H 2.6, C ) 5.0, D ) 5.4, E ) 5.9, F ) 6.5. Studies were carried out by incubation of myosin and cathepsin D as given in legend to Fig. 4a. Control studies were carried out by substitution of distilled water for the enzyme at each p H value investigatedfollowed by 24 h incubation. The control shown was performed at p H 6.5 and was representative of each control carried out at the various p H values studied.
626
Edward A. Ogunro, Richard B. Lannian, Julia
These results suggest a) that cathepsin D catalyses the degradation of myosin into specific end products; b) that the 18600 dalton light chain of myosin may be more susceptible to cleavage by cathepsin D than the 28000 dalton chain since the former exhibits a considerably reduced staining intensity following 24 h incubation with enzyme at pH 6.5. The apparent trend towards the formation of polypeptides of similar molecular weight following incubation at pH values between 2.6 and 5.9 (Fig. 4b) together with the considerable reduction in the rate of myosin hydrolysis (observed at pH 2.6) between 4 and 24 h compared with that observed between 10 min and 4 h (Fig. 4a) suggests two major possibilities: a) That cathepsin D may be unstable at these pH values and that the trend towards the formation of similar “end products” of myosin degradation may result from instability of the enzyme, or b) That the degradation products of myosin observed following incubation at pH 2.6 may be relatively resistant to further hydrolysis and thus may represent the end products of myosin degradation by this particular enzyme. In order to investigate these possibilities, the following experiments were undertaken: 1) The stability of cathepsin D was investigated by incubating the enzyme (37”c9 24 h, at various PH values, f o b w e d by measurement of the residual cathepsin D activity using haemoglobin (see Methods). Samples incubated at 4°C (24 h) served as controls for this study. 2) The ability of cathepsin D to catalyse further degradation of the polypeptides produced following incubation (pH 2.6, 24 h) of myosin with the enzyme was investigated by addition of a second aliquot of cathepsin D to samples of myosin which had previously been incubated with cathepsin D for 24 h. The results from the first study (Table) show that cathepsin D exhibits varying stability when incubated at different pH values. Of the pH values investigated, the enzyme was most stable at pH 5.4 and exhibited lower stability on either side of this pH Table The stability of cathepsin D at various p H . ~~
~
PH
% Control activity remaining after 24 h incubation
2.6 5.0 5.4 5.9 6.4
13.3 35.2 45.8 40.9 28.3
Results represent the mean of duplicate determinations.
R. Spencer, Alan G. Fergusotr. and Michael Lesrh
A
B
C
Fig. 5a Investigation of the ability of cathepsin D to catalyse complete degradation of myosin at p H 2.6. A ) o.25 mg myosin incubated for 48 h in the absence of cathepsin D (distilled water substituted for enzyme), B ) 0.25 mg myosin incubated with 6.25 vg cathepsin D for 24 h, C ) as in B with further addition of 6.25 pg carhepsin D at 24 h and additional 24 h incubation.
value. Following incubation at pH 2.6 only I3 % of the control activity was recovered, indicating that cathepsin D was relatively unstable under these conditions. However, since the results of experiments (Fig. 5a) in which a second aliquot of cathepsin D was added to myosin samples previously incubated at pH 2.6 for 24 h (experiment 2) failed to show further hydrolysis of the 49000 and 29000 dalton polypeptides observed at 24 h, the results suggest that these polypeptides may be resistant to further hydrolysis by cathepsin D and consequently may represent the “end products” of myosin degradation by this enzyme. b) Actin The results of experiments in which the degradation of actin by cathepsin D was monitored using SDS electrophoresis are shown in Fig. 5b. In contrast to the studies with myosin, the proteolysis of actin did not result in the progressive formation of polypeptides of decreasing molecular weight. However, the overall size of the actin band was considerably reduced at 24 h compared with the control.
621
Degradation of nivosin and actin by cathepsin D Inhibition studies with pepstatin A
In order to verify that the degradation of myosin and actin resulted from cathepsin D activity and not from contamination by another protease, studies were carried out in which the myofibrillar proteins were incubated with cathepsin D in the presence and absence of the acid protease inhibitor pepstatin A. Studies with myosin were carried out at pH values of 2.6 and 6.5 while studies with actin were carried out at pH 4.2. Pepstatin A completely inhibited the degradation of myosin at pH 2.6 and 6.5 (Fig. 6) and similar results were also obtained when actin was substituted for myosin in the incubation medium. These results indicate that the observed degradation of myosin and actin was mediated specifically by cathepsin D and show that cathepsin D not only catalyses the degradation of myofibrillar proteins at specific acid pH optima, but that the enzyme also catalyses degradation at pH values close to neutrality.
Discussion A
B
Fig. 5b The proteolysis of actin by cathepsin D. A ) Control, B ) cathepsin D + actin. Studies were carried out by incubation of 0.20 trig actin with 5.0 pg cathepsin D followed by 24 h incubationat pH 4.2. Control studies were carried out by substitution of distilled water for cathepsin D and incubation as in test samples.
The results presented in this paper show that canine cardiac cathepsin D readily catalyses the hydrolysis of myosin and actin isolated from homologous tissue. The pH optimum of the enzyme for myosin hydrolysis was highly acidic (pH 2.6, 3.4) and was distinctly different from that found to be optimal for actin hydrolysis (pH 4.2). Despite this highly acidic
Fig. 6 Recorder scan tracings of SDS gels showing inhibition of the cathepsin D mediated degradation of myosin at pH 6.5. Similar results were obtained at pH 2.6. SDS gels were scanned at 650 nm using the Beckman 576767 gel scanner accessory for the model 25 spectrophotometer (Beckman Instruments, lrvine, California). A ) Control, B ) cathepsiti D + myosin, C ) cathepsin D + myosin + pepstatin A . Studies were perjormed by incubation (24 h ) of 0.25 mg mvosin with 6.25 I L cathepsin ~ D in the presence or absence of pepstatin A (1.0 pmol.litre - 1 ) . Control studies were performed by substitution of distilled water for cathepsirr D jollowed by 24 h incubation. HC= myosin heavy chain, LC,=MW 28000 light chain, LC,= M W 18600 light chain. Note appearance in B ) of heavy chain degradation products and disappearance of LC,.
628
Edward A. Ogimro. Richard B. Lannwn, Julia
pH optimum however, cathepsin D catalysed the hydrolysis of myosin at near neutral pH (6.5). In the present study, the degradation of actin at pH 6.5 could not be investigated since the SDS electrophoretic assay procedure was not sufficiently sensitive. This lack of sensitivity resulted from the fact that smaller molecular weight degradation products of actin could not be detected following incubation with cathepsin D and subsequent SDS electrophoresis. The ability of cathepsin D to catalyse the degradation of myosin at pH values (6.5) significantly less acidic than the pH optimum of the enzyme suggests that the cathepsin D preparation may have been contaminated with proteases exhibiting pH optima less acidic than that of cathepsin D. However, this was unlikely since the degradation of myosin was completely inhibited by pepstatin A, a specific inhibitor of cathepsin D (Woessner, 1972). Thus although cathepsin D catalyses the hydrolysis of myosin at pH values greater than its optimum, the rate of hydrolysis is markedly reduced. This observation is in agreement with previous data from this laboratory (Ogunro et a/., 1979) which indicated a similar phenomenon with cathepsin D hydrolysis of myoglobin and haemoglobin. In these studies, Michaelis-Menten kinetics were used to analyse the effect of pH on the cathepsin D catalysed hydrolysis of myoglobin and haemoglobin at pH values less acidic (4.5) than the pH optimum of the enzyme. The results of these , of the cathepsin D restudies showed that V action was decreased while K, was unchanged (haemoglobin) or decreased (myoglobin) indicating a similar or more efficient binding of enzyme and substrate despite the lowered reaction rate. The results of incubation studies carried out at various pH values suggest that the cathepsin D mediated hydrolysis of myosin follows a particular pattern and terminates with the formation of specific polypeptide end products which appear to be relatively resistant to further extensive cleavage by cathepsin D. In this connection it is of interest to speculate that these polypeptides may represent the end products of myosin degradation by cathepsin D in vivo and that other intracellular proteases may be responsible for subsequent hydrolysis of the polypeptides into oligopeptides and amino acids. Previous reports concerning the degradation of myosin have shown that proteases such as trypsin (Perry, 1951) and chymotrypsin (Gergely, 1955) readily catalyse hydrolysis of this protein. However, while these studies have yielded valuable information regarding the structure of the myosin molecule (Lowey and Holtzer, 1959), they have provided little insight into the possible function of intracellular proteases in the degradation of myofibrillar pro-
R. Spencer, Alan C. Fergtison, and Michael Lesch
teins in muscle cells. In this context, the susceptibility of myosin and actin to cleavage by lysosomal acid proteases has not been fully defined. Although it has been suggested (Suzuki et a/., 1969; Schwartz and Bird, 1977) that myosin and actin are susceptible to cleavage by intracellular acid proteases, the majority of the studies carried out have implicated these enzymes specifically in the degradation of sarcoplasmic rather than myofibrillar proteins (Bodwell and Pearson, 1964; Drabikowski e / d., 1977). While the results of these latter studies may have been the consequence of a lack of sensitivity in the assay procedures employed, there are also criticisms associated with those studies which have demonstrated activity of cathepsin D towards myosin and actin. For example it is not clear from the studies by Suzuki e/ a/. (1969) and Schwartz and Bird (1977) whether homogeneous preparations of cathepsin D were employed, since no homogeneity criteria were presented by these authors. In the study by Schwartz and Bird (1977). the majority of experiments were performed with cathepsin D isolated from hepatic tissue, furthermore myosin light chains were not resolved by the electrophoretic procedure which these investigators employed and thus they were unable to comment on the susceptibility of the myosin light chains to cleavage by cathepsin D. In this connection the results obtained in the present study suggest that the MW 18600 light chain of myosin appears to be more susceptible to degradation by cathepsin D than the MW 28000 light chain. In conclusion, in contrast to the studies by Suzuki et a/. ( 1 969) and Schwartz and Bird ( I 977) we have demonstrated that cathepsin D not only catalyses the gross degradation of myosin and actin but that myosin is degraded into specific polypeptides which are relatively resistant to further extensive hydrolysis. We have also demonstrated that cathepsin D catalyses the hydrolysis of myosin at pH values as high as 6.5. These observations may be of considerable physiological significance since they suggest firstly that multiple protease enzyme systems may be responsible for the complete degradation of proteins in vivo and secondly that lysosomal acid proteases may not necessarily function at or close to their pH optima within the cell. Supported in part by USPHS grant No. NHLBI19648 and a grant from the Oppenheimer Family Foundation. References
L. (1938). The estimation of pepsin. trypsin and cathepsin with hemoglobin. Jorrrnal of Gmrrd Physiologj.
Anson, M .
22.19-89.
Degradation
of nijtosin and acrin by cathepsin D
Bodwell, C. E., and Pearson, A. M. (1964). The activity of partially purified bovine catheptic enzymes o n various natural and synthetic substrates. Journal of Food Science, 29,602-607. Drabikowski, W. (1961). The proteolytic activity of actin preparations. Acta Biochimica Polonica. 8, 3-14. Drabikowski, W., Gorecka, A,, Jakubiec-Puka. A. (1977). Endogenous proteinases in vertebrate skeletal muscle. International Journal of Biochemistry, 8, 61-7 I . Gergely, J., Gouvea, M. A., and Karibian, D. (1955). Fragmentation of myosin by chymotrypsin. Journal of Biological Chemistry. 212, 165-1 77. Griffin, W. S. T..and Wildenthal, K. (1978). Myofibrillar alkaline protease activity in rat heart and its responses t o some interventions which alter cardiac size. Journal of Molecular and Cellular Cardiology, 10, 669-676. Lowey, S., and Holtzer, A. (1959). The homogeneity and molecular weights of the meromyosins and their relative proportions in myosin. Biochimica et Biophjfrica Aria. 34, 470-484. Lowry. 0. H.,Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Marks. N., Grynbaum, A.. and Lajtha, A. (1973). Pentapeptide (pepstatin) inhibition of brain acid protease. Science. 181, 949-95 I . Mommaerts, W. F. H. M. (1958). Chemical investigation of muscular tissues. Methods in Medical Research. 7, 1-59. Morgan, H. E., Rannels, D. E., and Kao, R. L. (1974). Factors controlling protein turnover in heart muscle. Circulation Research. 34/35 Supplement Ill, 22-29. Murakami, K., and Inagami, T. (1975). Isolation of pure and stable renin from hog kidney. Biochemical and Biophysical Research Communications. 62, 757-763. Ohkuma, S.. and Poole, B. (1978). Fluorescence probe measurement of the intralysosomal pH in living cells and the peturbation of pH by various agents. ProceedinRs of the National Academy of Sciences, 75, 3327-333 I . Ogunro, E. A.. Ferguson. A. G., and Lesch. M. (1979). A kinetic study of the pH optimum of rabbit myocardial cathepsin D. Submitted to Cardiovavcular Research. Perry, S . V. (1951). The adenosine triphosphatase activity of myofibrils isolated from skeletal muscle. Biochemical Journal, 48,257-265. Radola, B. J. (1973). Analytical and preparative isoelectric focusing in gel stabilized layers. Annals of the New York Academy of Sciences. 209, 127-143. Reddy, M. K., Etlinger. J. D.. Rabinowitz, M., Fischman, D. A. (1975). Removal of 2 lines and a-actinin from isolated myofibrils by a calcium-activated neutral protease. Journal of BiOlcJgiCal Chemistry, 250,4278-4284.
62Y Rees, M. K., and Young, M. (1967). Studies on the isolation and molecular properties of homogeneous globular actin. Journal of Biological Chemistry. 242, 4449-4458. Reijngoud, D. J., and Tager, J. M. (1973). Measurement of lntralysosomal pH. Biuchiniica ('I Biuphysica Acta. 297, 174- 178. Riccuitti. M. A. (1972). Lysosomes and myocardial cellular injury. American Journal of Cardiology. 30, 492-497. Rice, R. H., and Means. G. E. (19711. Radioactive labelling of proteins in vitro. Journal of Biological Cliemistry, 246, 83 1-832. Sarkar, S., Sreter. F. A., and Gergely. J. (1971). Light chains of myosin from white, red and cardiac muscles. Proceedings of The National Acac1eni.v of Sciences. 5, 946-950. Schwartz, A. N., and Bird, J. W. C. (1977). Degradation of myofibrillar proteins by cathepsins B and D. Biochemical Journal, 167, 8 I 1-820. Sender, P. M. (1971). Muscle fibrils: solubilization and gel electrophoresis. FEBS letters, 17. 106-1 10. Shiverick, K. T.,Thomas, L. L.. and Alpert, N . R. (1975). Purification of cardiac myosin. Application to hypertrophied myocardium. Biochimica rt Biophysica Acta, 393, 124-1 33. Suzuki. A.. Okitani, A.. and Fujimaki, M. (1969). Studies on proteolysis in stored muscle. Part IV. Effect of cathepsin D treatment on ATPase activity of myosin B. Agricdtiiral Biological Chemistry. 33, 1723- 1729. Szent-Gyorgyi, A. (1947). Chrmistr,. of Miisciilar contraction. Academic Press, New York. Uchida, K.. Murakami. U., and Hiratsuka, T. (1977). Purification of cardiac myosin from rat heart: Proteolytic cleavage and its inhibition. Journal ofBiochemistry, 82,469476. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determinations by sulfate-polyacryl-amide gel electrophoresis, Journal of Biological Chrmistry. 244, 4406-44 12. Welman, E.. and Peters, T. J. (1977). Enhanced lysosome fragility in the anoxic perfused guinea pig heart: Effects of glucose and mannitol. Journal of Molrciclar and Cellular Cardiology. 9, I 0 1-I 20. Wildenthal, K. (1978). Lysosomal alterations in ischemic myocardium: result or cause of myocellular damage. Journal of Moleculw and Cellular Cardiology, 10, 595-603. Woessner. J. F. (1972). Pepstatin inhibits the digestion of hemoglobin and protein-polysaccharide complex by cathepsin D. Biochemical and Biuphi,.sical Research Contnumication.s.47, 965-970.
Degradation of canine cardiac myosin and actin by cathepsin D isolated from homologous tissue EDWARD A . O G U N R O , R I C H A R D B. LANMAN, J U L I A R. S P E N C E R , ALAN G . FERGUSON, A N D MICHAEL LESCH From the Department of’ Medicine, Section of Cardiology, Northwestern University Medical School, Chicago, Illinois
The ability of canine cardiac cathepsin D to catalyse hydrolysis of myosin andactin isolated from homologous tissue was monitored using both SDS polyacrylamide gel electrophoresis and the release of trichloroacetic acid soluble radioactivity from the radioactively (14C) labelled proteins. Cathepsin D readily catalysed the hydrolysis of myosin and actin. The pH-activity profile for myosin hydrolysis exhibited two optima with a major peak at pH 2.6 together with a smaller peak at pH 3.4. This was distinct from the pH-activity profile for actin hydrolysis where a single optimum at pH 4.2 was observed. Incubation of cathepsin D with myosin (pH 2.6, 37°C) resulted in complete degradation of myosin light chains (MW = 2 8 0 0 0 ~ 7 0 0and 18600k350, n = 4 ) within 10 min while heavy chains (MW= 190400f2500, n=4) were degraded into three proteins of molecular weight 32OOOi-750; 37000k1000 and 49000k750 (n-4) within 4 h. Further incubation (24 h) of cathepsin D with myosin resulted in two proteins of MW 29OOOk2000 and 48800k3100 (n=4). In the case of actin (MW 44800k 1600, n=6) polypeptides produced as a result of hydrolytic cleavage by cathepsin D (pH 4.2, 37°C) were not observed on SDS gels, although the overall size of the actin band was markedly decreased at 24 h. Cathepsin D catalysed the hydrolysis of myosin at near neutral pH (6.5) although the overall rate of degradation was considerably reduced compared to that observed at pH 2.6. Pepstatin A was a potent inhibitor of the hydrolysis of myosin and actin at all pH values investigated indicating that cathepsin D and not a contaminant was responsible for the observed degradation.
SUMMARY
Cardiac lysosomal acid proteases are thought to play an essential role in cell death following ischaemic injury (Ricciutti, 1972; Welman and Peters, 1977; Wildenthal, 1978) and in the turnover of myocardial proteins (Morgan et al., 1974). At the present time however, the specific role of these enzymes in the above degradative processes is incompletely defined. This arises as a consequence of a number of reasons. Firstly, little data is available on the susceptability of major myocardial proteins to hydrolysis by lysosomal acid proteases.Secondly, many acid proteases exhibit pH optima that are considerably lower than the pH values thought to exist within the lysosome (Reijngoud and Tager, 1973; Ohkuma and Poole, 1978). These observations together with reports of the presence of myocardial tissue proteases with neutral (Reddy et al., 1975) and alkaline (Griffin and Wildenthal, 1978) pH optima, have focused conReprint requests to: Edward A. Ogunro, Ph.D., Department
of Medicine, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, Illinois 6061 I , USA.
siderable attention upon the need to define the role of lysosomal acid proteases in mammalian myocardial protein catabolism. The aim of the studies reported in this paper were twofold: 1) To study whether cathepsin D (EC 3.4.4.23, a major lysosomal acid protease) isolated from myocardial tissue catalyses the degradation of myofibrillar proteins such as myosin and actin isolated from homologous tissue. 2) To investigate whether the isolated cathepsin D is capable of catalysing the hydrolysis of myosin or actin at pH values close to physiological pH. Materials and methods All reagents were of the highest analytical grade commercially available and were obtained from either Fisher Scientific, Itasca, Illinois; Scientific Products, McGaw Park, Illinois, or The Sigma Chemical Company, St. Louis, Missouri. Radioactively labelled chemicals were obtained from New England Nuclear, Boston, Massachusetts. 62 1
622
Edward A. Ogunro, Richuril B. Latinrun, Jiilia H. Spmwr, Alun ti. Fwgusorr, untl Micliud L i w l i
PURIFICATION OF CATHEPSIN D Cathepsin D was purified approximately 2500 fold to homogeneity from canine cardiac tissue using techniques previously developed (Ogunro et a/., 1979) for the isolation of this enzyme from rabbit myocardium. Hearts were removed from dogs by performing a right thoracotomy following chloralose anaesthesia. The hearts were washed with sodium chloride (1 50 mmol.litre-l) to remove blood, trimmed, blotted dry, minced, and finally homogenised for eight periods of 15 s at 15 s intervals (Waring Blender, top speed, 4 C) in three volumes of a solution containing sodium chloride ( I 50 mmol. litre-]) and butanol (270 mmol*litre-I). Triton X-100 was added to the homogenate to give a final concentration of 0.1 % w/v (in order to maximise disruption of lysosomes) and the homogenate was stirred (30 min) and centrifuged at 7000 g for 20 min. The pellet (PI) was discarded and the supernatant (S1) was further centrifuged at IOOOOO g for 60 min. The pellet (obtained following 1OOOOO g centrifugation, P,) was discarded and the supernatant (S,)was dialysed for 12 h against acetate buffer (100 mmol. litre-], pH 3.7) containing sodium chloride (500 mrnol*litre-') in order to remove the butanol and Triton X-100. During dialysis some protein precipitation occurred and this was removed by centrifugation at IOOOOO g for 15 rnin. The supernatant (S,) from this latter centrifugation was filtered through a 10 pm filter (Millipore Corporation, Bedford, Massachusetts) and concentrated to approximately 10 cms. The concentrated solution (50 to 60 mg p r o t e i n a ~ - ~was ) run onto a pepstatinylsepharose affinity column prepared according to the procedure of Murakami and lnagami (1975) and was continuously washed with sodium acetate buffer (100 mmol*litre-l, pH 3.7 NaCl 500 mmol.litre-l until the absorbance (280 nm) of the column effluent was less than 0.05. The eluting buffer was then changed to bicarbonate (100 mmol.litre-l, pH 8.3 /NaCI 50 mmol.litre-l) and 10 fractions of 5 cms volume were collected. The fractions with highest specific activity of acid protease were pooled, concentrated to approximately 1 cms (6 to 12 mg protein*~m-~) and further purified by gel filtration chromatography (sephacryl S200) using the above bicarbonate buffer as eluant. Twenty fractions of 3 cms volume were collected. The fractions with the highest specific activity of acid protease were again pooled, concentrated to a volume of 2 cms (3 to 6 mg p r o t e i n a r a ) dialysed against deionised water for 12 h (two changes) and finally subjected to isoelectric focusing (Radola, 1973) using pH gradient of 3.5 to 10.0. During isoelectric focusing, major cathepsin D activity equilibrated between pH values of 7.0 and 7.6 and these fractions were pooled and
stored (pH 7.0 to 7.6) at -20 C. Under these conditions the enzyme was stable for periods up to 4 months. PURIFICATION OF MYOSIN
Myosin was purified from canine cardiac tissue according to the procedure of Shiverick et a/. ( 1975) with the exception that ATP (2.5 mmol.litre-l) was added to the myosin homogenisation - extraction solution to facilitate the dissociation of actomyosin and thereby increase the yield of myosin (Mommaerts, 1958). PURIFICATION OF G-ACTIN The procedure of Szent-Gyorgyi ( 1947) was used to prepare an acetone powder from canine myocardium. Subsequent steps in the purification procedure involved extraction of G-actin from the acetone powder, polymerisation to form F-actin, depolymerisation and gel filtration using sephadex G-200 (Rees and Young, 1967). RADIOACTIVE LABELLING OF MYOSIN AND ACTIN
Myofibrillar proteins were radioactively (I4C) labelled using the reductive alkylation procedure of Rice and Means (1971). Following labelling, protein solutions were dialysed to remove radioactive byproducts. Solutions of myosin were dialysed (40 h, 3 changes) against imidazole buffer ( 10 mmol-litre-l, pH 6.8) containing KCI (0.5 mol.litre-l) and dithiothreitol (2.0 mmol.litre-l) while solutions of actin were similarly dialysed against an ATP solution (0.5 mmol-litre-', pH 7.5) which contained 2mercaptoethanol (0.5 mmol*litre-l) and CaCI, (0.2 mmol*litre-I). In general, solutions of labelled myosin were freshly prepared for each experiment, however actin samples were stored (4 'C) for periods of up to 2 weeks. PROTEIN DETERMINATIONS
Two methods of protein determination were used. In studies with myosin, the ratio of absorbance at 280 nm and 260 nm was employed. In studies with actin the procedure of Lowry et a/. (1951) was used with crystalline human serum albumin as standard. EVALUATION OF MYOSIN A N D ACTIN AS SUBSTRATES FOR CATHEPSIN D
Two assay procedures were employed : 1) Quantitation of the release of trichloroacetic acid (TCA) soluble radioactivity from radioactively labelled myosin and actin. 2) Analysis of degradation products of myosin and actin using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. This method
623
Degradarion of myosin and actin hv cathepsin D
was employed in addition to the radioactive procedure since it offered the possibility of a greatly improved assay sensitivity in situations where large (TCA insoluble) polypeptides may have been produced following protease action. In the first method, radioactively labelled myofibrillar protein and cathepsin D were incubated (37'C, 30 min) at a ratio of 70:1 w/w respectively. The reaction mixture (0.2 cms) contained myosin (0.20to 0.25 mg) or actin (0.20to 0.25 mg), cathepsin D, sodium acetate buffer (0.2 mol-litre-', pH 2.0 to 7.0) and KCI (0.5mol*litre-l). Following incubation, the reaction was terminated by precipitation of all proteins with 0.2cm3TCA (0.6mol*litre-') and the assay mixture was further incubated (10 min) at 0°C to ensure complete precipitation. The samples were centrifuged (lOO000 g x 10 min) in a microcentrifuge (Beckman Instruments, Irvine, California) and the radioactivity in 0.1 cm3 aliquots of the supernatant fraction was determined (Packard Tricarb Scintillation Counter Model 3375,Packard Instrument Company, Inc., Downers Grove, Illinois) using 10 cms Instagel Scintillation mixture (Packard Instrument Company, Inc.). In the second method non radioactively labelled myofibrillar protein and cathepsin D were incubated (37"C,10 min to 24 h) as described above, however the ratio of myofibrillar protein to cathepsin D was increased to 4O:l w/w to improve sensitivity for detection of degradation products of myosin or actin. Following incubation the reaction was terminated by cooling the mixture to 0°C. Samples containing myosin were dialysed against a solution of imidazole (10 mmol.litre-l, pH 6.8) containing KCI (0.5 mol*Iitre-l), while samples of actin were dialysed against a solution of ATP (0.5mmol*litre-l, pH 7.5) containing 2-mercaptoethanol (0.5 mollitre-') and CaC12 (0.2 mmol.litre-'). SDS electrophoresis was subsequently performed using the method of Weber and Osborn (1969).In both assay procedures blank determinations were carried out in which distilled water was substituted for cathepsin D. Since there has been some indication of protease activity associated with actin preparations (Drabikowski, 1961),all actin samples were heated to 100°C for 10 min before use in order to inactivate such enzyme activity. Sodium acetate buffer was employed throughout the pH ranges investigated in order to minimise possible differential effects of various buffer solutions on the pH-activity profile for cathepsin D. N o detectable change in pH was observed during the incubation period at any of the pH values investigated. D ACTIVITY Cathepsin D was assayed as described by Anson ASSAY OF CATHEPSIN
(1938).
MOLECULAR WEIGHT DETERMINATIONS
Molecular weight determinations using the SDS electrophoretic technique of Weber and Osborn (1969)were performed on the purified preparations of myosin and actin and on the degradation products of these proteins following incubation with cathepsin D. Two groups of standards were employed : 1) High molecular weight standards: Myosin heavy chain (MW =200000), galactosidase ( 1 3oooO), phosphorylase B (940001, bovine serum albumin (68000) and ovalbumin (43000).These proteins were obtained as a kit from Bio-Rad, Laboratories Inc., Richmond, California. 2) Low molecular weight standards: Human serum albumin (68000), pepsinogen (43000),a chymotrypsinogen A (25500) and cytochrome C (12380).
Results EVALUATION OF MYOSIN A N D ACTIN PREPARATIONS
Myosin and actin were typically isolated from canine cardiac tissue with yields of 5 m g p and 1 mg.gl tissue wet weight respectively. The Anea:Aoso ratio of the isolated myosin was usually greater than 1.45 and the specific activity of the radioactively labelled proteins was of the order of 1 x loe dpmmg-I protein for myosin and 1 x lo5 d p m m g l protein for actin. On SDS polyacrylamide gels the purified myosin was represented by a single heavy chain of molecular weight 190400f2500, n=4, a number of minor proteins of molecular weight approximately 12oooO and two light chains of MW 28000 f700 and 18600 h350 n =4. SDS electrophoresis of purified actin revealed the presence of a single polypeptide of molecular weight 44800 f1600, n =6. The values calculated for the molecular weights of myosin heavy and light chains and actin were in good agreement with values previously published for the molecular weights of these proteins isolated from cardiac (Sarkar et a/., 1971 ; Uchida et a/.,1977)and other tissues (Rees and Young, 1967;Sender, 1971). EVALUATION OF CATHEPSIN PREPARATION
D
SDS electrophoretic studies of cathepsin D isolated from canine myocardial tissue revealed the presence of two polypeptides of molecular weight approximately 32000 and 14OOO daltons (Fig. 1). The smaller polypeptide accounted for approximately 20% of the total protein and probably represented a subunit of the enzyme since it was not observed following polyacrylamide gel electrophoresis performed in the absence of sodium dodecyl sulphate.
Edward A. Ogunro, Richard B. Lanman, Julia R. Spencer, Alan G . Ferguson. and Michael Lesch
624
myosin hydrolysis (Fig. 3a) was bimodal with a major optimum at pH 2.6 together with a smaller peak (approximately 80% of the first peak) at pH 3.4. These results were distinctly different from the pH-activity profile for the hydrolysis of actin (Fig. 3b) where a single optimum at a pH of approximately 4.2 was observed. 2) Studies with unlabelledproteins a ) Myosin The cathepsin D mediated degradation of
Fig. 1 Typical results obtainedfollowing SDS electrophoresis (under reducing conditions) of the purified canine cardiac cathepsin D. E V A L U A T I O N OF M Y O S I N A N D A C T I N AS SUBSTRATES FOR CATHEPSIN D
1) Studies with labelled proteins Cathepsin D readily catalysed the degradation of myosin and actin as verified by the progressive release of TCA soluble radioactive peptides from the radioactively labelled proteins (Fig. 2). pH optimum studies revealed that the pH-activity profile for
--
a) M Y O S W
b) ACTIN
w 60 -
Fig. 2 Linearity of a ) myosin and b) actin hydrolysis by cathepsin D using the radioactively labelled myofibrillar proteins as substrates. Results represent the mean of duplicate determinations. Studies were carried out by incubation of 0.20 to 0.25 mg of myosin or actin (2.9 to 3.6 pg cathepsin D) at p H values of 2.6 and 4.2 respectively. Control studies were carried out by substituting distilled water for the enzyme in 1 -. L - - 1 -_Idthe assay mixture followed by 60 20 40 60 80 100 niin (myosin) or 100 min (actin) incubation, INCUBATION TIME ( m m )
120 -
too
INCUBATION TIME (min )
myosin at pH 2.6 (as monitored using SDS electrophoresis) is shown in Fig. 4a. Myosin light chains were completely degraded within 10 min of incubation while heavy chains were degraded into proteins of molecular weight 32000f750; 37000 t lo00 and 490003~750daltons (n=4) within 4 h. Further incubation at this pH (up to 24 h) resulted in a negligible change in molecular weight of the 49000 dalton protein, however the 37000 and 32000 dalton proteins were further degraded and appeared as a diffuse protein band of MW 29000+2000 (n=4). The results of studies in which the hydrolytic activity of cathepsin D towards myosin was compared at pH values ranging from’2.6 to 6.5 are shown in Fig. 4b. Cathepsin D catalysed the degradation of myosin at all the pH values investigated, however the overall rate of hydrolysis was considerably reduced as the pH of the incubation medium was increased to 6.5. At pH 5.0 myosin was degraded within 24 h into three major proteins of molecular weight 33000 1500; 40000 3000 and 49000flSOO (n=4). It is evident from Fig. 6 that polypeptides of similar molecular weight are also produced following incubation at pH values of 5.4 and 5.9. At pH 6.5 however, the overall rate of degradation is considerably reduced such that the formation of these polypeptides can not be seen at 24h.
625
DeRradation of ni.vosin and actin by cathepsin D _ _ ~
._
a) MYOSIN
b) ACTIN
~
2o O2.0
t
6.0 01 20
3.0L 4.0 - 5.0 pH OF ASSAY
I
I
I
5.0 pH OF ASSAY
3.0
4.0
A A
B
C
D
L, 6.0
B
Fig. 3 pH-activity profiles for cathepsin D using radioactively labelled a) myosin and b) actin as substrates. Results represent the mean of duplicate determinations. Studies were carried out by incubation ofmyosin or actin and cathepsin D, as in legend to Fig. 2. at the appropriate p H values followed by 30 min incubation. Control studies were performed by substitution of distilled water for the enzyme at each p H value investigated followed by a similar periodof incubation.
C
D
E
F
E
Fig.4a Typical results obtained following incubation of cathepsin D with myosin at p H 2.6 and subsequent SDS electrophoresis (under reducing conditions). A) Control. B ) 10 min, C ) 30 min, D ) 4 h and E ) 24 h incubation. Studies were carried out by incubating 0.20 to 0.25 nig of myosin with 5.0 to 6.5 pg cathepsin D for the indicated time period. Control studies were performed 6.v substitution of distilled water for the enzyme followed by 24 h incubation.
Fig. 4b Typical results obtained following incubation (24 h) of cathepsin D and myosin at various p H values. A ) Control, B ) p H 2.6, C ) 5.0, D ) 5.4, E ) 5.9, F ) 6.5. Studies were carried out by incubation of myosin and cathepsin D as given in legend to Fig. 4a. Control studies were carried out by substitution of distilled water for the enzyme at each p H value investigatedfollowed by 24 h incubation. The control shown was performed at p H 6.5 and was representative of each control carried out at the various p H values studied.
626
Edward A. Ogunro, Richard B. Lannian, Julia
These results suggest a) that cathepsin D catalyses the degradation of myosin into specific end products; b) that the 18600 dalton light chain of myosin may be more susceptible to cleavage by cathepsin D than the 28000 dalton chain since the former exhibits a considerably reduced staining intensity following 24 h incubation with enzyme at pH 6.5. The apparent trend towards the formation of polypeptides of similar molecular weight following incubation at pH values between 2.6 and 5.9 (Fig. 4b) together with the considerable reduction in the rate of myosin hydrolysis (observed at pH 2.6) between 4 and 24 h compared with that observed between 10 min and 4 h (Fig. 4a) suggests two major possibilities: a) That cathepsin D may be unstable at these pH values and that the trend towards the formation of similar “end products” of myosin degradation may result from instability of the enzyme, or b) That the degradation products of myosin observed following incubation at pH 2.6 may be relatively resistant to further hydrolysis and thus may represent the end products of myosin degradation by this particular enzyme. In order to investigate these possibilities, the following experiments were undertaken: 1) The stability of cathepsin D was investigated by incubating the enzyme (37”c9 24 h, at various PH values, f o b w e d by measurement of the residual cathepsin D activity using haemoglobin (see Methods). Samples incubated at 4°C (24 h) served as controls for this study. 2) The ability of cathepsin D to catalyse further degradation of the polypeptides produced following incubation (pH 2.6, 24 h) of myosin with the enzyme was investigated by addition of a second aliquot of cathepsin D to samples of myosin which had previously been incubated with cathepsin D for 24 h. The results from the first study (Table) show that cathepsin D exhibits varying stability when incubated at different pH values. Of the pH values investigated, the enzyme was most stable at pH 5.4 and exhibited lower stability on either side of this pH Table The stability of cathepsin D at various p H . ~~
~
PH
% Control activity remaining after 24 h incubation
2.6 5.0 5.4 5.9 6.4
13.3 35.2 45.8 40.9 28.3
Results represent the mean of duplicate determinations.
R. Spencer, Alan G. Fergusotr. and Michael Lesrh
A
B
C
Fig. 5a Investigation of the ability of cathepsin D to catalyse complete degradation of myosin at p H 2.6. A ) o.25 mg myosin incubated for 48 h in the absence of cathepsin D (distilled water substituted for enzyme), B ) 0.25 mg myosin incubated with 6.25 vg cathepsin D for 24 h, C ) as in B with further addition of 6.25 pg carhepsin D at 24 h and additional 24 h incubation.
value. Following incubation at pH 2.6 only I3 % of the control activity was recovered, indicating that cathepsin D was relatively unstable under these conditions. However, since the results of experiments (Fig. 5a) in which a second aliquot of cathepsin D was added to myosin samples previously incubated at pH 2.6 for 24 h (experiment 2) failed to show further hydrolysis of the 49000 and 29000 dalton polypeptides observed at 24 h, the results suggest that these polypeptides may be resistant to further hydrolysis by cathepsin D and consequently may represent the “end products” of myosin degradation by this enzyme. b) Actin The results of experiments in which the degradation of actin by cathepsin D was monitored using SDS electrophoresis are shown in Fig. 5b. In contrast to the studies with myosin, the proteolysis of actin did not result in the progressive formation of polypeptides of decreasing molecular weight. However, the overall size of the actin band was considerably reduced at 24 h compared with the control.
621
Degradation of nivosin and actin by cathepsin D Inhibition studies with pepstatin A
In order to verify that the degradation of myosin and actin resulted from cathepsin D activity and not from contamination by another protease, studies were carried out in which the myofibrillar proteins were incubated with cathepsin D in the presence and absence of the acid protease inhibitor pepstatin A. Studies with myosin were carried out at pH values of 2.6 and 6.5 while studies with actin were carried out at pH 4.2. Pepstatin A completely inhibited the degradation of myosin at pH 2.6 and 6.5 (Fig. 6) and similar results were also obtained when actin was substituted for myosin in the incubation medium. These results indicate that the observed degradation of myosin and actin was mediated specifically by cathepsin D and show that cathepsin D not only catalyses the degradation of myofibrillar proteins at specific acid pH optima, but that the enzyme also catalyses degradation at pH values close to neutrality.
Discussion A
B
Fig. 5b The proteolysis of actin by cathepsin D. A ) Control, B ) cathepsin D + actin. Studies were carried out by incubation of 0.20 trig actin with 5.0 pg cathepsin D followed by 24 h incubationat pH 4.2. Control studies were carried out by substitution of distilled water for cathepsin D and incubation as in test samples.
The results presented in this paper show that canine cardiac cathepsin D readily catalyses the hydrolysis of myosin and actin isolated from homologous tissue. The pH optimum of the enzyme for myosin hydrolysis was highly acidic (pH 2.6, 3.4) and was distinctly different from that found to be optimal for actin hydrolysis (pH 4.2). Despite this highly acidic
Fig. 6 Recorder scan tracings of SDS gels showing inhibition of the cathepsin D mediated degradation of myosin at pH 6.5. Similar results were obtained at pH 2.6. SDS gels were scanned at 650 nm using the Beckman 576767 gel scanner accessory for the model 25 spectrophotometer (Beckman Instruments, lrvine, California). A ) Control, B ) cathepsiti D + myosin, C ) cathepsin D + myosin + pepstatin A . Studies were perjormed by incubation (24 h ) of 0.25 mg mvosin with 6.25 I L cathepsin ~ D in the presence or absence of pepstatin A (1.0 pmol.litre - 1 ) . Control studies were performed by substitution of distilled water for cathepsirr D jollowed by 24 h incubation. HC= myosin heavy chain, LC,=MW 28000 light chain, LC,= M W 18600 light chain. Note appearance in B ) of heavy chain degradation products and disappearance of LC,.
628
Edward A. Ogimro. Richard B. Lannwn, Julia
pH optimum however, cathepsin D catalysed the hydrolysis of myosin at near neutral pH (6.5). In the present study, the degradation of actin at pH 6.5 could not be investigated since the SDS electrophoretic assay procedure was not sufficiently sensitive. This lack of sensitivity resulted from the fact that smaller molecular weight degradation products of actin could not be detected following incubation with cathepsin D and subsequent SDS electrophoresis. The ability of cathepsin D to catalyse the degradation of myosin at pH values (6.5) significantly less acidic than the pH optimum of the enzyme suggests that the cathepsin D preparation may have been contaminated with proteases exhibiting pH optima less acidic than that of cathepsin D. However, this was unlikely since the degradation of myosin was completely inhibited by pepstatin A, a specific inhibitor of cathepsin D (Woessner, 1972). Thus although cathepsin D catalyses the hydrolysis of myosin at pH values greater than its optimum, the rate of hydrolysis is markedly reduced. This observation is in agreement with previous data from this laboratory (Ogunro et a/., 1979) which indicated a similar phenomenon with cathepsin D hydrolysis of myoglobin and haemoglobin. In these studies, Michaelis-Menten kinetics were used to analyse the effect of pH on the cathepsin D catalysed hydrolysis of myoglobin and haemoglobin at pH values less acidic (4.5) than the pH optimum of the enzyme. The results of these , of the cathepsin D restudies showed that V action was decreased while K, was unchanged (haemoglobin) or decreased (myoglobin) indicating a similar or more efficient binding of enzyme and substrate despite the lowered reaction rate. The results of incubation studies carried out at various pH values suggest that the cathepsin D mediated hydrolysis of myosin follows a particular pattern and terminates with the formation of specific polypeptide end products which appear to be relatively resistant to further extensive cleavage by cathepsin D. In this connection it is of interest to speculate that these polypeptides may represent the end products of myosin degradation by cathepsin D in vivo and that other intracellular proteases may be responsible for subsequent hydrolysis of the polypeptides into oligopeptides and amino acids. Previous reports concerning the degradation of myosin have shown that proteases such as trypsin (Perry, 1951) and chymotrypsin (Gergely, 1955) readily catalyse hydrolysis of this protein. However, while these studies have yielded valuable information regarding the structure of the myosin molecule (Lowey and Holtzer, 1959), they have provided little insight into the possible function of intracellular proteases in the degradation of myofibrillar pro-
R. Spencer, Alan C. Fergtison, and Michael Lesch
teins in muscle cells. In this context, the susceptibility of myosin and actin to cleavage by lysosomal acid proteases has not been fully defined. Although it has been suggested (Suzuki et a/., 1969; Schwartz and Bird, 1977) that myosin and actin are susceptible to cleavage by intracellular acid proteases, the majority of the studies carried out have implicated these enzymes specifically in the degradation of sarcoplasmic rather than myofibrillar proteins (Bodwell and Pearson, 1964; Drabikowski e / d., 1977). While the results of these latter studies may have been the consequence of a lack of sensitivity in the assay procedures employed, there are also criticisms associated with those studies which have demonstrated activity of cathepsin D towards myosin and actin. For example it is not clear from the studies by Suzuki e/ a/. (1969) and Schwartz and Bird (1977) whether homogeneous preparations of cathepsin D were employed, since no homogeneity criteria were presented by these authors. In the study by Schwartz and Bird (1977). the majority of experiments were performed with cathepsin D isolated from hepatic tissue, furthermore myosin light chains were not resolved by the electrophoretic procedure which these investigators employed and thus they were unable to comment on the susceptibility of the myosin light chains to cleavage by cathepsin D. In this connection the results obtained in the present study suggest that the MW 18600 light chain of myosin appears to be more susceptible to degradation by cathepsin D than the MW 28000 light chain. In conclusion, in contrast to the studies by Suzuki et a/. ( 1 969) and Schwartz and Bird ( I 977) we have demonstrated that cathepsin D not only catalyses the gross degradation of myosin and actin but that myosin is degraded into specific polypeptides which are relatively resistant to further extensive hydrolysis. We have also demonstrated that cathepsin D catalyses the hydrolysis of myosin at pH values as high as 6.5. These observations may be of considerable physiological significance since they suggest firstly that multiple protease enzyme systems may be responsible for the complete degradation of proteins in vivo and secondly that lysosomal acid proteases may not necessarily function at or close to their pH optima within the cell. Supported in part by USPHS grant No. NHLBI19648 and a grant from the Oppenheimer Family Foundation. References
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Anson, M .
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Degradation
of nijtosin and acrin by cathepsin D
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