Biochimica et Biophysica Acta, 1121 (1992)47-53
47
~ 1992 Elsevier Science Publishers B.V. All rights reserved f)167-4838/92/Yd15.00
BBAPRO 34189
Polycional antisera specific for the proenzyme form of each calpain Dorothy E. Croall a, Clive A. Slaughter b, Helen S. Wortham ~, Colleen M. Skelly ~', Lynn DeOgny b and Carolyn R. Moomaw ~ "Departmenl of Biochemi.~try, Micr~dTiolog~'and Molecular Bioloks', Ttze Unirersit~"of Maine, Orono, ME (USA) and h The ltoward lhzghes Medical Institutes, Unit'ersity c~fTexas StJuthwe~tcrn Medical Center, Dallas, TX [USA)
(Received 3 September 1991)
Key words: Calpain; Proteinase; Proteolysis; Immunoblolting Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes arc cxpo~d to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. Wc have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-(erminal epitope of milli-calpain was u ~ d to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitopc of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of tt-calpain in rat erythrocytes treated with ionomycin and calcium.
Introduction
Recen'. evidence from several laboratories suggests that the ubiquitous, intracellular, calcium-activated neutral proteinases (CANPs, EC 3.4.22.17; also known as calpains or ealcium-d~;pendent proteinases) are heterodimeric, proenzymes until Ca 2+-binding occurs and autoproteolysis of the N-terminus of each subunit produces active enzymes that exhibit increased .sensitivity to calcium ions [1-5]. This model for enzyme activation is based on studies of the purified proteinases [1-5], but requires additional confirmation of the functional significance of specific autoproteolytic cleavage events [6-8] and requires demonstration of its significance in vivo. The regulatory subunit is identical for both calpains [7,8] and undergoes a significant change in molecular weight after proteolytic modification (from 30-17 kDa; Refs. 1-3). We have previously used polyclonal antibodies raised against the milli-calpain holoenzyme to document proteolytic modification of the regulatory subunit of both proteinases [9]. We observed increased proteolytic modification of the micro-calpain regulatory subunit in rat erythrocytes in response to incubation with ionomycin and calcium.
Correspondence: D.E. Croall, 277 Hitehner ltall, Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469, USA.
Although analysis of the regulatory subunit provides evidence for the activation of each calpain, this approach can not discriminate between the two enzymes, micro-calpain (p. or type-l) and milli-calpain (m- or type-2), in this report we describe synthetic peptides, containing sequences from the amino terminal region of each unique, catalytic subunit, that were used as antigens and the characteristics of the polyclonal antisera raised against them. Materials and Methods
Each calpain was purified from bovine heart muscle using previously published methods [10]. Proteolytically modified micro-caipain was generated in vitro by incubation with 5.77 mM CaCI~ in the presence of (I.61 mM EGTA and 0.61 mM EDTA at 4°C for 2 min. Autoproteolysis was terminated by dilution with EGTA (25 mM) and heating for 10 min at 65°C in the presence of SDS-PAGE sample buffer. The calpain subunits were separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes [11] (Millipore, Bradford, MA) and the amino terminal sequence of each modified subunit was determined by automated Edman degradation using an Applied Biosystems Model 670A sequenator. These sequences are shown in Table I. Adjacent N-terminal sequences are taken from the human cDNA-derived sequence published by Aoki et al. [12]. The comparable se-
48 TABLE !
Autoproteolytic prot'csdng sitt's within micro, arm milli-calpabt The amino-terminal protein sequences determined for bovine pcalpain 78 kDa and 17 kDa proteins are underlined. The amino terminal peplide ~quence o[ autoproteolytically modified m-calpain is taken from imajoh et a]. [13.14]. Sequences preceding each cleavage site are derived from published eDNA ~quences fi)r human p-calpain 84 kDa [12]. human m-calpain 811 kDa [i3] and I~wine m-calpain 26 kDa [20]. 84/78 kDa p-calpain 26/17 kDa/z-calpain 80/80 kDa m-calpain
VQKQRARELG LGRHENAIKY PRTHYSNIE AAKLAKDREAAEGLG
ANESEEVRQF SHERAIKY
quences from milli-calpain, as published previously by others [13,14] are shown for comparison. Based on these sequences, the following peptides were synthesized using solid-phase, tBoc-chemistry in an Applied Biosystems Model 430A peptide synthesizer according to the manufacturers standard conditions: CAAVQKQRARELGLGRHENAIKY and C A A K L A K DREAAEGLGSHERA. The pcptidcs were conjugated to keyhole limpet hemocyanin (Sigma) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) according to the method described by Mumby and Gilman [15]. PeptideKLH conjugates were mixed with Freund's complete (first injection) or Freund's incomplete (all subsequent injections) adjuvant and injected sub-cutaneously in multiple sites for each rabbit (New Zealand White). Serum was prepared from clotted blood and heat-inactivated (56°C for 30 rain). This crude serum was the :~ource of antibodies for immunoblots. immunoblots were prepared by electrophoretic transfer of proteins from SDS-polyacrylamide gels to nitrocellulose membranes (Schleicher and Schueli, BA85) as previously described [9]. Standard transfer conditions were in 25 mM Tris-192 mM glycine (pH 8.3), 20% methanol for 4 h at 170 mA and 80 mA overnight. For all blots, incubation of the primary ~ n , with the immobilized antigen was for 3 h at room temperature and reactive material was visualized using alkaline-phosphatase conjugated goat anti-rabbit IgG and the color-producing substrates, toluidine p-nitro tetrazolium blue (NBT) and 5-bromo-4 chloro-3 indolyl phosphate (BCIP) were purchased from Bio-Rad. The relative efficiencies of electrophoretic transfer of the unmodified and proteolyzed 80 kDa subunits were determined by comparisons of immunoblots probed with antisera raised against the holo-enzymes. For mcalpain there appeared to be little difference in the efficiency of transfer of the proteolyzed or non-proteolyzed 80 kDa subunit. The immunoreactive signal for the proteolyzed 80 kDa was 86% (mean; range 74106%) that obtained with an equal amount of the unproteolyzed subunit. For p-calpain the autoprote-
olyzed subunit was either reduced in transfer efficiency or has lost more immunoreactivity after autoproteolysis. The immunoreactive signal for the autoproteolyzed catalytic subunit was 72% (mean; range 63-80%) that of an equivalent amount of unproteolyzed 84 kDa. An LKB-Pharmacia UItroscan LX was used for densitometric analysis of stained gels and immunoblots. Proteolytie activity was measured using ~4C-methylated casein (60%-70a, Sigma C7891) as substrate, under conditions described in our earlier studies [3,8, 17]. Protein concentrations were estimated by the method of Bradford [18] using commercially prepared reagent (Bio-Rad Laboratories) and bovine serum albumin as standard. Isolation of rat erythrocytes and the conditions used for incubation with ionomycin and calcium were as described previously [9]. Details of the samples analyzed in this report are given in Fig. 7. Results
Antibody specificity. The anti-peptide antisera were characterized using each purified calpain in both the proenzyme and the proteolyticaily modified, calciumactivated forms (Fig. 1). It is essential that the epitope-specific antibodies are highly specific for only one of the two enzymes to allow us to discriminate 1
2
3
4
80_
-.
.
. i
26_
.
.
',::
r :.
Q -
~:•:k•!•-::: !rrI
Fig. 1. Coomassie blue R-250 stained SDS-PAGE of/z-calpain and m-calpain: proenzyme and proteolytically modified forms. Both calpains were purified from bovine heart in their proenzyme forms {p.-calpain, lane 2 and m-calpain, lane 4). Auloprotcolysis was stimulated by adding calcium chloride (2 mM for tz-calpain and 7 mM for m-calpain) to the enz'~rnes as described in Materials and Methods. Autoproteolysis was allowed to proceed at 4°C for/~-calpain for 1 rain (lane I) and at 25°C for m-calpain for 2.5 rain (lane 3). Autoproteolysis was terminated with the addition of SDS sample buffer containing EGTA and heating. 5 pg of protein was loaded on each lane.
49 between micro-calpain and milli-calpain in samples that contain both enzymes. Results of immunoblot experiments that demonstrate the specificity of each antipeptide antibody for the proenzymc form of its parent proteinasc are shown in Figs. 2 and 3. Densitometric analysis of these and similar blotting experiments demonstrates that each antiserum has high selectivity for the unmodified form of only one catalytic subunit (F,.'g. 3); despite the presence of 15-50% of the synthetic peptide sequence in the activated, proteolytically modified proteins. There was no demonstrable cross-reactivity of either antisera against the heterologous enzyme (even at 2 /zg enzyme blotted per lane, data not shown), although 11/18 residueg of the mcalpain peptide are identical to the/z-calpain peptide. Proteolytic modification of the catalytic subunits. To determine if the N-terminal peptide is removed from the catalytic subunit of m-c.alpain prior to, or concomitant with, casein hydrolysis and to further examine if this modification is necessary for expression of proteolytic activity against the casein substrate, the following series of experiments was done. Milli-calpain was preincubated with calcium under conditions that allow autoproteolysis to occur at a relatively slow rate. At various times, samples were diluted in EGTA containing buffers and either treated with SD,¢ for electrophoresis and immunoblotting or added to a buffered solution of radiolabelled casein to assay proteolytic activity. Assay conditions for measuring casein hydrolyA.
a bcde
fg
sis were chosen to minimize additional autoproteolysis of the added calpain (sub-optimal calcium concentration and lower temperature). Results from such experiments are shown in Figs. 4-6. For the casein hydrolysis assays (Fig. 4) the total calpain protein added to each assay was identical. Thus these data demonstrate that preincubation alters calpain in some way that enhances proteolytic activity. However, if casein hydrolysis was measured at 22°C in the presence of 7 mM calcium chloride (conditions known to allow rapid conversion of the calpain to its proteolytically modified form during the assay for enzyme activity), short, preincubation times did not alter the rate or amount of casein hydrolyzed (data not shown). The epitope-directed antiserum specific for m-calpain was used to examine the autoproteolytic modification of the catalytic subunit in comparison with the total calpain catalytic subunit as measured by Coomassie blue staining or immunoblots probed with antisera raised against the ho[oenzymes (Fig. 5A and B). Densitometric quantification of immunobiots of the N-terminal epitope of the catalytic subunit are shown in Fig. 6. The combined data from the experiments described in Figs. 4-6 thus document an inverse relationship between the initial apparent rate of casein hydrolysis and the amount of the Nterminal peptide still present within the catalytic subunit. The regulatory subunit of calpain was also proteolyrically modified during the preincubation of the proteinascs with calcium (data not shown). This result is
h I | k Iron
C.
a bcdef r:~--~ ~.....
gh
l j k
!
':-~~ - 8 4 .: :~:i/~~?~,~.~,"
: ,';.:~ .
B.
D. •. 2 6 26
..17 17
Fig. 2. Specificity and sensitivity of polyclonal antisera raised against N-terminal pcptides of m/cro-calpain and miLli--calpain. Panels A-D: immunoblots of unmodified and autoproteolyzed calpains. Samples from Fig. 1 were diluted with SDS-PAGE sample buffer for electrophoresis and transferred to nitrocellulose membranes, In panel A, p.-calpain proenzyme (15, 30, 50, 100, 150 or 200 ng; lanes i-n), or autoproteolyzed /z-calpain (15, 30, 50, 100, 200, 300, 400 rig; lanes g-a) were analyzed for their ability to react with antiserum raised against the #-calpain N-terminal peptide. The proenzyme form of m-calpain (200 rig) was analyzed in lane h. Antiserum was used at I : 1500 dilution. In panel C samples of m-calpain procnzym¢ (25, 50, 100, 15tJor 200 ng: lanes g-k) or autoproteolyzed m-calpain (25, 50, 100, 150, 200 or 300 ng; lanes f-a) were electrophoresed and blotted. Lane 1 contained 200 ng ,u.-calpain. The blot was incubated with a ! : 1000 dilution of the antiserum raised against the m-calpain terminal peptide. Panels B and D, show the lower portion of each blot described for A and C. These were incubated with polyclonal antiserum raised against m-calpain hotocnzyme 0 / 4 0 0 dilution) and reveal the 26 kDa subunit of the enzymes a n d / o r its proteolytic fragments in the autoprotcolyzed samples. The immunoreactive bands between 26 and 17 kDa (panel D) are fragments of the 26 kDa subunit because the autoproteolyzed m-calpain used for this experiment had not completely converted the 26 kDa to its 17 kDa form [3,9,20].
50 consistent with our earlier studies [3]. Similar experiments with micro-calpain demonstrate increased initial rates of casein hydrolysis with increasing time of preincubation of this enzyme with calcium and the concomitant loss of the N-terminal epitope of the 84 kDa catalytic subunit (data not shown).
Proteolytic modi#cation of the micro-calpain catalytic subunit in rat erythrocytes. For bovine micro-calpain, modification of both the catalytic and regulatory subunits is detectable by SDS-PAGE. Blots probed with the anti-peptide antiserum confirmed that the amino terminal epitope is removed from the catalytic subunit during proteolytic processing to the 78 kDa form. Although the micro-calpain catalytic subunit from bovine (or human) samples undergoes a shift in apparent molecular weight that is detected by SDS-PAGE, the micro-calpain from rat tissues does not [3,6,8,19]. The epitope-specific antiserum demonstrates the proteolytic modification of this subunit in the absence of a shift in relative mobility. Whole cell iysates, prepared from erythrocytes treated with ionomycin in the pres-
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47
~ 1992 Elsevier Science Publishers B.V. All rights reserved f)167-4838/92/Yd15.00
BBAPRO 34189
Polycional antisera specific for the proenzyme form of each calpain Dorothy E. Croall a, Clive A. Slaughter b, Helen S. Wortham ~, Colleen M. Skelly ~', Lynn DeOgny b and Carolyn R. Moomaw ~ "Departmenl of Biochemi.~try, Micr~dTiolog~'and Molecular Bioloks', Ttze Unirersit~"of Maine, Orono, ME (USA) and h The ltoward lhzghes Medical Institutes, Unit'ersity c~fTexas StJuthwe~tcrn Medical Center, Dallas, TX [USA)
(Received 3 September 1991)
Key words: Calpain; Proteinase; Proteolysis; Immunoblolting Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes arc cxpo~d to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. Wc have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-(erminal epitope of milli-calpain was u ~ d to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitopc of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of tt-calpain in rat erythrocytes treated with ionomycin and calcium.
Introduction
Recen'. evidence from several laboratories suggests that the ubiquitous, intracellular, calcium-activated neutral proteinases (CANPs, EC 3.4.22.17; also known as calpains or ealcium-d~;pendent proteinases) are heterodimeric, proenzymes until Ca 2+-binding occurs and autoproteolysis of the N-terminus of each subunit produces active enzymes that exhibit increased .sensitivity to calcium ions [1-5]. This model for enzyme activation is based on studies of the purified proteinases [1-5], but requires additional confirmation of the functional significance of specific autoproteolytic cleavage events [6-8] and requires demonstration of its significance in vivo. The regulatory subunit is identical for both calpains [7,8] and undergoes a significant change in molecular weight after proteolytic modification (from 30-17 kDa; Refs. 1-3). We have previously used polyclonal antibodies raised against the milli-calpain holoenzyme to document proteolytic modification of the regulatory subunit of both proteinases [9]. We observed increased proteolytic modification of the micro-calpain regulatory subunit in rat erythrocytes in response to incubation with ionomycin and calcium.
Correspondence: D.E. Croall, 277 Hitehner ltall, Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469, USA.
Although analysis of the regulatory subunit provides evidence for the activation of each calpain, this approach can not discriminate between the two enzymes, micro-calpain (p. or type-l) and milli-calpain (m- or type-2), in this report we describe synthetic peptides, containing sequences from the amino terminal region of each unique, catalytic subunit, that were used as antigens and the characteristics of the polyclonal antisera raised against them. Materials and Methods
Each calpain was purified from bovine heart muscle using previously published methods [10]. Proteolytically modified micro-caipain was generated in vitro by incubation with 5.77 mM CaCI~ in the presence of (I.61 mM EGTA and 0.61 mM EDTA at 4°C for 2 min. Autoproteolysis was terminated by dilution with EGTA (25 mM) and heating for 10 min at 65°C in the presence of SDS-PAGE sample buffer. The calpain subunits were separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes [11] (Millipore, Bradford, MA) and the amino terminal sequence of each modified subunit was determined by automated Edman degradation using an Applied Biosystems Model 670A sequenator. These sequences are shown in Table I. Adjacent N-terminal sequences are taken from the human cDNA-derived sequence published by Aoki et al. [12]. The comparable se-
48 TABLE !
Autoproteolytic prot'csdng sitt's within micro, arm milli-calpabt The amino-terminal protein sequences determined for bovine pcalpain 78 kDa and 17 kDa proteins are underlined. The amino terminal peplide ~quence o[ autoproteolytically modified m-calpain is taken from imajoh et a]. [13.14]. Sequences preceding each cleavage site are derived from published eDNA ~quences fi)r human p-calpain 84 kDa [12]. human m-calpain 811 kDa [i3] and I~wine m-calpain 26 kDa [20]. 84/78 kDa p-calpain 26/17 kDa/z-calpain 80/80 kDa m-calpain
VQKQRARELG LGRHENAIKY PRTHYSNIE AAKLAKDREAAEGLG
ANESEEVRQF SHERAIKY
quences from milli-calpain, as published previously by others [13,14] are shown for comparison. Based on these sequences, the following peptides were synthesized using solid-phase, tBoc-chemistry in an Applied Biosystems Model 430A peptide synthesizer according to the manufacturers standard conditions: CAAVQKQRARELGLGRHENAIKY and C A A K L A K DREAAEGLGSHERA. The pcptidcs were conjugated to keyhole limpet hemocyanin (Sigma) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) according to the method described by Mumby and Gilman [15]. PeptideKLH conjugates were mixed with Freund's complete (first injection) or Freund's incomplete (all subsequent injections) adjuvant and injected sub-cutaneously in multiple sites for each rabbit (New Zealand White). Serum was prepared from clotted blood and heat-inactivated (56°C for 30 rain). This crude serum was the :~ource of antibodies for immunoblots. immunoblots were prepared by electrophoretic transfer of proteins from SDS-polyacrylamide gels to nitrocellulose membranes (Schleicher and Schueli, BA85) as previously described [9]. Standard transfer conditions were in 25 mM Tris-192 mM glycine (pH 8.3), 20% methanol for 4 h at 170 mA and 80 mA overnight. For all blots, incubation of the primary ~ n , with the immobilized antigen was for 3 h at room temperature and reactive material was visualized using alkaline-phosphatase conjugated goat anti-rabbit IgG and the color-producing substrates, toluidine p-nitro tetrazolium blue (NBT) and 5-bromo-4 chloro-3 indolyl phosphate (BCIP) were purchased from Bio-Rad. The relative efficiencies of electrophoretic transfer of the unmodified and proteolyzed 80 kDa subunits were determined by comparisons of immunoblots probed with antisera raised against the holo-enzymes. For mcalpain there appeared to be little difference in the efficiency of transfer of the proteolyzed or non-proteolyzed 80 kDa subunit. The immunoreactive signal for the proteolyzed 80 kDa was 86% (mean; range 74106%) that obtained with an equal amount of the unproteolyzed subunit. For p-calpain the autoprote-
olyzed subunit was either reduced in transfer efficiency or has lost more immunoreactivity after autoproteolysis. The immunoreactive signal for the autoproteolyzed catalytic subunit was 72% (mean; range 63-80%) that of an equivalent amount of unproteolyzed 84 kDa. An LKB-Pharmacia UItroscan LX was used for densitometric analysis of stained gels and immunoblots. Proteolytie activity was measured using ~4C-methylated casein (60%-70a, Sigma C7891) as substrate, under conditions described in our earlier studies [3,8, 17]. Protein concentrations were estimated by the method of Bradford [18] using commercially prepared reagent (Bio-Rad Laboratories) and bovine serum albumin as standard. Isolation of rat erythrocytes and the conditions used for incubation with ionomycin and calcium were as described previously [9]. Details of the samples analyzed in this report are given in Fig. 7. Results
Antibody specificity. The anti-peptide antisera were characterized using each purified calpain in both the proenzyme and the proteolyticaily modified, calciumactivated forms (Fig. 1). It is essential that the epitope-specific antibodies are highly specific for only one of the two enzymes to allow us to discriminate 1
2
3
4
80_
-.
.
. i
26_
.
.
',::
r :.
Q -
~:•:k•!•-::: !rrI
Fig. 1. Coomassie blue R-250 stained SDS-PAGE of/z-calpain and m-calpain: proenzyme and proteolytically modified forms. Both calpains were purified from bovine heart in their proenzyme forms {p.-calpain, lane 2 and m-calpain, lane 4). Auloprotcolysis was stimulated by adding calcium chloride (2 mM for tz-calpain and 7 mM for m-calpain) to the enz'~rnes as described in Materials and Methods. Autoproteolysis was allowed to proceed at 4°C for/~-calpain for 1 rain (lane I) and at 25°C for m-calpain for 2.5 rain (lane 3). Autoproteolysis was terminated with the addition of SDS sample buffer containing EGTA and heating. 5 pg of protein was loaded on each lane.
49 between micro-calpain and milli-calpain in samples that contain both enzymes. Results of immunoblot experiments that demonstrate the specificity of each antipeptide antibody for the proenzymc form of its parent proteinasc are shown in Figs. 2 and 3. Densitometric analysis of these and similar blotting experiments demonstrates that each antiserum has high selectivity for the unmodified form of only one catalytic subunit (F,.'g. 3); despite the presence of 15-50% of the synthetic peptide sequence in the activated, proteolytically modified proteins. There was no demonstrable cross-reactivity of either antisera against the heterologous enzyme (even at 2 /zg enzyme blotted per lane, data not shown), although 11/18 residueg of the mcalpain peptide are identical to the/z-calpain peptide. Proteolytic modification of the catalytic subunits. To determine if the N-terminal peptide is removed from the catalytic subunit of m-c.alpain prior to, or concomitant with, casein hydrolysis and to further examine if this modification is necessary for expression of proteolytic activity against the casein substrate, the following series of experiments was done. Milli-calpain was preincubated with calcium under conditions that allow autoproteolysis to occur at a relatively slow rate. At various times, samples were diluted in EGTA containing buffers and either treated with SD,¢ for electrophoresis and immunoblotting or added to a buffered solution of radiolabelled casein to assay proteolytic activity. Assay conditions for measuring casein hydrolyA.
a bcde
fg
sis were chosen to minimize additional autoproteolysis of the added calpain (sub-optimal calcium concentration and lower temperature). Results from such experiments are shown in Figs. 4-6. For the casein hydrolysis assays (Fig. 4) the total calpain protein added to each assay was identical. Thus these data demonstrate that preincubation alters calpain in some way that enhances proteolytic activity. However, if casein hydrolysis was measured at 22°C in the presence of 7 mM calcium chloride (conditions known to allow rapid conversion of the calpain to its proteolytically modified form during the assay for enzyme activity), short, preincubation times did not alter the rate or amount of casein hydrolyzed (data not shown). The epitope-directed antiserum specific for m-calpain was used to examine the autoproteolytic modification of the catalytic subunit in comparison with the total calpain catalytic subunit as measured by Coomassie blue staining or immunoblots probed with antisera raised against the ho[oenzymes (Fig. 5A and B). Densitometric quantification of immunobiots of the N-terminal epitope of the catalytic subunit are shown in Fig. 6. The combined data from the experiments described in Figs. 4-6 thus document an inverse relationship between the initial apparent rate of casein hydrolysis and the amount of the Nterminal peptide still present within the catalytic subunit. The regulatory subunit of calpain was also proteolyrically modified during the preincubation of the proteinascs with calcium (data not shown). This result is
h I | k Iron
C.
a bcdef r:~--~ ~.....
gh
l j k
!
':-~~ - 8 4 .: :~:i/~~?~,~.~,"
: ,';.:~ .
B.
D. •. 2 6 26
..17 17
Fig. 2. Specificity and sensitivity of polyclonal antisera raised against N-terminal pcptides of m/cro-calpain and miLli--calpain. Panels A-D: immunoblots of unmodified and autoproteolyzed calpains. Samples from Fig. 1 were diluted with SDS-PAGE sample buffer for electrophoresis and transferred to nitrocellulose membranes, In panel A, p.-calpain proenzyme (15, 30, 50, 100, 150 or 200 ng; lanes i-n), or autoproteolyzed /z-calpain (15, 30, 50, 100, 200, 300, 400 rig; lanes g-a) were analyzed for their ability to react with antiserum raised against the #-calpain N-terminal peptide. The proenzyme form of m-calpain (200 rig) was analyzed in lane h. Antiserum was used at I : 1500 dilution. In panel C samples of m-calpain procnzym¢ (25, 50, 100, 15tJor 200 ng: lanes g-k) or autoproteolyzed m-calpain (25, 50, 100, 150, 200 or 300 ng; lanes f-a) were electrophoresed and blotted. Lane 1 contained 200 ng ,u.-calpain. The blot was incubated with a ! : 1000 dilution of the antiserum raised against the m-calpain terminal peptide. Panels B and D, show the lower portion of each blot described for A and C. These were incubated with polyclonal antiserum raised against m-calpain hotocnzyme 0 / 4 0 0 dilution) and reveal the 26 kDa subunit of the enzymes a n d / o r its proteolytic fragments in the autoprotcolyzed samples. The immunoreactive bands between 26 and 17 kDa (panel D) are fragments of the 26 kDa subunit because the autoproteolyzed m-calpain used for this experiment had not completely converted the 26 kDa to its 17 kDa form [3,9,20].
50 consistent with our earlier studies [3]. Similar experiments with micro-calpain demonstrate increased initial rates of casein hydrolysis with increasing time of preincubation of this enzyme with calcium and the concomitant loss of the N-terminal epitope of the 84 kDa catalytic subunit (data not shown).
Proteolytic modi#cation of the micro-calpain catalytic subunit in rat erythrocytes. For bovine micro-calpain, modification of both the catalytic and regulatory subunits is detectable by SDS-PAGE. Blots probed with the anti-peptide antiserum confirmed that the amino terminal epitope is removed from the catalytic subunit during proteolytic processing to the 78 kDa form. Although the micro-calpain catalytic subunit from bovine (or human) samples undergoes a shift in apparent molecular weight that is detected by SDS-PAGE, the micro-calpain from rat tissues does not [3,6,8,19]. The epitope-specific antiserum demonstrates the proteolytic modification of this subunit in the absence of a shift in relative mobility. Whole cell iysates, prepared from erythrocytes treated with ionomycin in the pres-
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