- Email: [email protected]
Association of calpain with insoluble pellet of rat lens
Exp. Eye Res. (1992) 55. 369-375
Association KIRSTEN Departments
of Calpain J. LAMPI”,
of Biochemistry
(Received
Norwich
with
LARRY
Insoluble
L. DAVID
AND
Pellet THOMAS
of Rat Lens R. SHEARER
and Ophthalmology, Schools of Dentistry and Medicine, Sciences University, Portland, OR 97201, U.S.A. 78 September
7997 and accepted
in revised form 4 November
Oregon Health
7997)
Calpains are calcium-activated neutral proteases found in many tissues including the lens, The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated Into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of
calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates : and (2) it may activate calpain, since membrane phospholipids lower the protease’s calcium requirement. Key words: calpain ; lens ; calcium ; membrane : calpastatin.
1. Introduction Calpains (CANP, EC 3 .4.22 .17) are cysteine proteases
with neutral pH optima and a requirement for calcium activation (Murachi, 1984). They are found ubiquitously in animal tissues. Calpain II, requiring mM calcium,
is the predominant
form in the lens, and it
has been assayed in several different species. However, the overall concentration of free calcium in normal cells is not high enough to activate calpain II. Data from several laboratories have shown that the calcium activation requirement of calpain was reduced in the or substrates presence of specific phospholipids (Melloni et al., 1984; Pontremoli et al., 1985). It is hypothesized that in the presence of calcium levels below those required for catalytic activity, calpain may associate through an N-terminal hydrophobic domain with phospholipids in the cell membrane (Gopalakrishna and Barsky, 1986; Suzuki et al., 1988). At the membrane, calpain then becomes activated at calcium concentrations below those required for activation of soluble calpain (Suzuki et al., 1988). This may also localize the site of action of calpain and modulate the types of substrates available to it. Nothing is known about the subcellular distribution of calpain in lens fiber cells. The purpose of the present study was to determine if the activation mechanism postulated above may exist in the lens by: (1) measuring
the amount
and activity
of insoluble
* For reprint requests at: Department of Biochemistry, Oregon Health Sciences University, 611 SW. Campus Drive, Portland, OR 97201. U.S.A. 0014-4835/92/080369+07
calpain in the lens; and (2) determining the effect of calcium on the association of calpain with various fractions of the insoluble pellet. We present evidence here that while calpain was predominantly cytosolic in rat lens fiber cells, calpain was also associated with the membrane-rich fraction.
$0%00/O
2. Materials and Methods Animals and Materials
Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Lenses were dissected from enucleated eyes, decapsulated, and the whole lens used for the studies below. Antibody against calpain II was prepared in rabbits by injecting rat muscle calpain II. Calpastatin was purified from bovine heart muscle (Mellgren et al., 1988). Synthetic calpain inhibitors, E64 and a peptide aldehyde CbzValPheH, were obtained from Sigma and as a gift from Merrell-Dow Research Institute (Cincinnati, OH), respectively. isolation of Calpain in Subcelldar Fractions Lenses from 24-day-old rats were homogenized in buffer A containing 20 mM Tris, 1 mM EGTA, 1 mM EDTA, 1 mM DTE, 100 mM KCI, and 2 mM phenylmethylsulfonylfluoride (PMSF), pH 7.5 and centrifuged at 10000 g for 30 min. The insoluble pellet was washed twice in buffer A and then extracted in buffer B, which contained 7 M urea in 50 mM Tris and 1 mM EGTA (pH 7.4) (Russell, Robinson and Kinoshita. 198 1). The urea-insoluble pellet was resuspended in 0.1 M NaOH containing 10 mM /3-mercaptoethanol. 0 1992 Academic Press Limited
K. J. LAMPI
370
-
-
Lanes:
I
2
3
4
5
6
7
6
9
(0
1. Immunoblot for calpain in various fractions oflenses from 24-day-old rats. Arrows indicate SO- and 30 kDa subunits of calpain. Lane 1, molecular weight standards; 2, total water-soluble protein: 3. 7 M urea wash of insoluble pellet; 4, 0.1 M NaOH wash of urea-insoluble pellet: 5. remaining insoluble protein. All lanes contained 5Opug protein. Lanes 6-10 contain 2.5, 5, IO, 25, and 50 ng calpain standards. FIG.
Following
centrifugation the remaining insoluble were dissolved in 1% SDS. The above procedure was repeated with 15 9-week-old rats in which one lens from each rat was pooled and treated as above and the other lens processed with 1 mM CaCI, in all the buffers. E64 was included in the initial homogenizing buffer to prevent rapid degradation of calpain in the high calcium buffer. Membrane-rich fractions were obtained using sucrose gradients. Young 13-day-old rat lenses were homogenized in buffer C, which contained 5 mM Tris. 5 mM ,J-mercaptoethanol, 1 mM EGTA, 05 mM PMSF, and 10 ,u~ E64, pH 8.0 (Kistler et al., 1986). The homogenate was split in half and 2 mM CaCl, was added to one of the halves. The insoiuble pellet was isolated by centrifugation at 10000 g for 20 min and washed three times in the same buffer. The washed pellet containing 0.6 mg protein was suspended in 8 % sucrose (by weight) in buffer C and layered into a centrifuge tube loaded with 25, 45, 50 and 55% sucrose solutions for a total volume of 5 ml. The gradient was centrifuged in a Beckman SW 50.1 rotor head (Beckman. Inc. San Francisco, CA) for 90 min at 35 000 rpm. The material at the interfaces was collected, diluted four-fold in buffer C, and recentrifuged for 30 min at 100000 g. The pellets obtained from each interface were resuspended in buffer B and analysed for protein, phosphate, calpain and membrane protein MP26. Phosphate and MP26 were used as membrane markers. Protein was measured using proteins
ET AL.
the BioRad protein assay (BioRad. Richmond. CA). Phosphate content was determined with FiskeSubbarow reagent according to manufacturer’s directions (Sigma. St Louis, MO) and involved an organic extraction in chloroform : methanol ( 1: 1) followed by perchloric acid digestion. MP26 was identified from Coomassie blue stains of SDS-PAGE gels. Calpain was identified by immunoblotting as described below. Measurement of Calcium Dependency and Reversibility of Calpain Insolubilization The calcium dependency for binding of calpain to insoluble pellet was determined by homogenizing lenses from S-week-old rats in the presence of increasing amounts of added calcium (0 to 1 mM). The calpain inhibitor peptide CbzValPheH was added at 0.24 mM to prevent autolysis and calpain was quantitated by immunoblotting. CbzValPheH was chosen because it was more readily available in our laboratory than E64 at the time this experiment was conducted. Calpain activity and immunoreactivity were measured in water-soluble, chelator-extractable, and Triton X-100 solubilized proteins from 200 rat Ienses. age 24 days. Homogenization was carried out in 40 ml of 20 mM Tris (pH 7.51, 100 ,u~ EGTA, 2 mM DTE and 0.5 mM PMSF at 4°C. The homogenate was split in half and 200 /LM CaCl, was added to one portion. This represents both the EGTA-bound and -free calcium. The pellets were washed three times in the appropriate buffer. Calpain was extracted from the total insoluble pellets by homogenization in buffer A. without 100 mM KC1 three times. This calpain was designated ‘chelator-extract,able ‘. The remaining pellets were extracted overnight in 1% Triton X-100 followed by sonication. Triton was then removed using Pierce Extracti-gel (Pierce, Rockford, IL). All samples were applied to a TSK DEAE S-PW column and proteins eluted with a linear gradient of 0 to 0.5 M NaCl in buffer A. Calpain activity was assayed using FITClabeled casein (David and Shearer, 1986). One unit of calpain activity equaled 1 fig FITC-labeled, acidsoluble fragments released per min. Enzyme-linked immunosorbent assays @LISA) on DEAE fractions were formed using calpain II antibody. Electrophoresis and lmmunoblotting SDS-PAGE of various subcellular fractions was performed using 12 % acrylamide gels (Laemlli, 19 70). Proteins were then electrophoretically transferred to PVDF membrane (Millipore. Bedford, MA) using the Towbin buffer system (Towbin, Staehelin and Gordan, 19 79). Blots were incubated with anticalpain antibody and developed by alkaline phosphatase as recommended by the manufacturer (BioRad, Richmond, CA). Intensity was estimated by densitometry using a Hoefer GS-300 densitometer (Hoefer Scientific Instruments, San Francisco, CA). The amount of calpain in
CALPAIN
AND
INSOLUBLE
PELLET
371
TABLE I Effect of calcium on association of calpain with various lens fractions from g-week-old Homogenization in EGTA /lg calpain per mg protein
Subcellular fraction
CaCI,
% of total calpain
fig calpain per mg protein
% of total calpain
90.5 3.2 5.4 0.9 9.5
042 + 0.06* 2.09kO.22 0.97kO.09 1~90+0~10
61.7 19.6 15.9 2.8 38.3
1.00 +0.05* 0.59kO.07 0.42 + 0.10 0.93f0.20
Water-soluble Urea-soluble NaOH-soluble Pellet Total insoluble
Homogenization in 1 rn~
rats
* Meanks.D. 01= 5).
EGTA
Calcium
I
97
I
1
I
kDo
66
45
31
-
,MP
26
22
14
Lanes:
I
2
3
4
5
6
7
a
9
IO
FIG. 2. SDS-PAGE gel of 13-day-old rat lens insoluble pellet stained with Coomassie blue showing MP26 at the interfaces from a discontinuous sucrose gradient. The same volume was applied to each lane from identically treated samples. Lanes 1, 2, 3 and 4 are the g/25%, 25/45%, 45/50% and 50/55% sucrose interfaces, respectively. Lanes 6, 7, 8 and 9 are the same interfaces from a sucrose gradient in which 2 mM calcium was added to all the buffers, including the initial homogenate. Lanes 5 and 10 are from the pellets of the above gradients. unknown samples was calculated from a standard curve produced by adding known amounts of rat muscle calpain to the same immunoblot containing the unknown samples.
3. Results Distribution
of Calpain in Various Fractions of Lens
Calpain was found in all lens fractions isolated (Fig. 1). An immunoreactive band corresponding to the
80-kDa subunit of calpain was found in the watersoluble (cytosolic), urea-soluble (calpain easily dissociated from pellet), NaOH-soluble (calpain tightly bound to pellet), and SDS-soluble fractions (calpain possibly imbedded within membrane). Based on the densitometric measurements of the 80-kDa band, the majority of calpain, 94.2% was located in the cytosolic fraction when lenses were homogenized in the absence of calcium (Fig. 1). Significant portions of lens calpain were also loosely and tightly bound to the insoluble pellet. Relative amounts of 5.0%, 07 % 24-2
K. J.
372
ET AL.
TABLE II
and 0.1 y0 of total calpain were found in the urea-, NaOH- and SDS-extracted fractions (Fig. 1). The
Distribution of phosphatesin discontinuous sucrose
total protein isolated from the same four fractions was 88.8 mg of water-soluble protein, 6.5 mg of ureasoluble protein, 1.3 mg of NaOH-extracted protein, and 0.4 mg protein remaining in the insoluble pellet. The percentage of protein obtained in each fraction was similar to the percentages of calpain in each fraction. When lenses were homogenized in 1 mM CaCl,, calpain was redistributed from the cytosol into all fractions of the insoluble pellet (Table I). Individual insoluble fractions showed calcium-induced increases in calpain ranging from three- to six-fold. Insoluble calpain increased from 9.5% of the total lens calpain to 38.3% due to the addition of calcium, with the greatest increase in the urea-soluble fraction. These increases in the insoluble fraction were accompanied by a 29% decrease in the amount of total watersoluble calpain. The loss of soluble calpain was due to translocation of calpain to the insoluble pellet in the presence of calcium. This loss of soluble calpain was not due to a generalized insolubilization of lens proteins. The amount of insoluble protein did not increase. There was 6.2 mg of total insoluble protein from lenses homogenized in EGTA buffer and 56 mg of protein from lenses homogenized in calcium buffer. Insoluble calpain, however, increased from 3.1 to 7.9 ,ug. This indicated that the calcium-induced effect was specific for calpain. A greater percentage of insoluble calpain was present in 9-week-old lenses than in 24-day-old lenses (9.5 compared with 5*8x, respectively). As expected,
gradient
Interface
Homogenizationin EGTA nmol phosphates per j/g protein
Homogenizationin 1 mM CaCl, nmol phosphates per jig protein
S/25% 25145% 45/50X 50/55% Pellet
0.0 0.17 0.72 0.72 0.1 I
04 O,h7 0.76 (bl 3 0.2 3
total insoluble lens protein was larger in older rats than in younger ones (17.4 compared with 8.4x, respectively). Insoluble calpain and insoluble protein in the lens have been documented to increase with age (Takeana and Takemoto, 198 7 : Varnum, David and Shearer, 1989). The mechanism for the age-related calcium-induced insolubilization of calpain was not examined in this study. To obtain a more native membrane preparation, without the use of urea or NaOH, the insoluble pellet was separated on a discontinuous sucrose gradient. Material at the 25/45X, 45150% and 50155% interfaces was found to contain MP26 as the major protein (Fig. 2, lanes 2, 3,4). Phosphates, a marker for phospholipids, were also found at these interfaces (Table II). Some MP26 and phosphates pelleted at the bottom of the gradient (Fig. 2, lane 5; Table II).
EGTA
Calcium
Standards I
97
LAMPI
I
I
kDo
t
60 kDO
66
43
31
I
2
3
4
5
6
?
6
9
IO
II
12
I3
14
FIG.3. Immunoblot for calpainin 13-day-oldrat lensinsolublepelletfrom the interfacesof a discontinuoussucrosegradient. Seelegendto Fig. 2 for descriptionof lanes l-10. Lanes11, 12, 13. and 14 are 2.5, 5. 15 and 30 ng of calpain standards. respectively.
CALPAIN
AND
INSOLUBLE
PELLET
373
fractions as marked by MP26 and phosphates, and calcium increased this association. At the same time that calcium increased calpain association with the membrane, an absence of proteins above 3 1 kDa was observed (Fig. 2, lanes 7-9), and a decrease and shifting of bands was noted (Fig. 2, lane 10). This indicated that the concentration of E64 in these studies was not high enough completely to inhibit proteolysis by calpain or that other calcium proteases may exist in the lens. Calcium also altered the distribution of phosphates. The significance of this is unclear. Calcium Dependencyof the Association of Calpain with the InsoIubIePellet pt.4 calcium
FIG. 4. Calcium dependency curve for binding of calpain to lens insolnble pellet from 5-week-old rats. Each dot is an average of four determinations, with an average standard error of the mean = + 0.18 ,ug mg-I. Amount of calpain in pellet at 500 ,LLM calcium was greater than with 0 added calcium (P > 0.05).
3oI
To determine the calcium dependency for binding of calpain to the insoluble fraction, lenses from S-weekold rats were homogenized in buffers containing increasing amounts of calcium. With increasing calcium, more calpain became associated with the insoluble pellet (Fig. 4). The most dramatic increase in the insoluble calpain was seen when added calcium was elevated from 0 to 50 PM. The amount of calpain associating with pellet started to plateau at approximately 500 ,UM and a final three-fold increase over starting levels was observed at 1000 ,BM calcium. Reversibility of Calpainlnsolubilization
Calpain association with the insoluble pellet due to calcium was reversible, and the resolubilized enzyme remained active. Lenses were homogenized with and without 200 PM-free CaCl,, and the resulting insoluble pellets washed with high-chelator buffer. Lenses were homogenized at a calcium level chosen to increase insoluble calpain without causing extensive autolysis
FIG. 5. Specific amount of calpain in chelator [lane (B)] and Triton X-100 [lane (C)] washes of the insoluble pellet compared to water-soluble calpain [lane (A)]. q , Control values. q , Values from lenses homogenized in 200,uM
CaCl,. Lenses were from 24-day-old rats. Immunoblots showed calpain to be present in the same interfaces where MP26 was observed (Fig. 3, lanes 2, 3, 4). Calpain was not present at the 8/25X interface (Fig. 3, lane 1) and a dark-staining band of calpain was located at the bottom of the 55 % layer (Fig. 3, lane 5). This latter dense calpain was apparently not associated with lipid component. Adding calcium at a final concentration of 1 mM to the homogenizing buffer increased calpain found at the 25/45x, 45/50X and !iO/SS% interfaces (Fig. 3, lanes 7, 8, 9). Calcium did not alter the amount of MP26 found in these fractions. Calcium also increased the amount of calpain which pelleted (Fig. 3, lane 10). Thus. calpain was associatedwith the membrane-rich
and degradation of calpain. Four times more calpain was extracted from the pellet isolated in the presence of calcium than from the control pellet (Fig. 5). Calpain bound to the pellet due to calcium was resolubilized with chelator washes. The specific activity of this chelator-extractable calpain was 0.153 U mg-‘. This was greater than the water-soluble calpain activity (0.011 U mg-‘). The measurable activity was due to calpain because it eluted off a DEAE column at a salt concentration characteristic of calpain (David and Shearer, 1986). and it was completely inhibited by 100 ,UM E64, a synthetic cysteine protease inhibitor. The active fractions were also immunoreactive with calpain antibody in an ELISA assay. Some of the insolubilized calpain remained insoluble after several chelator washes and was only removed by Triton (Fig. 5). The activity of this Triton-solubilized calpain was 0105 U mg-‘. Calpain extracted with Triton from the insoluble pellets of lenses homogenized without added calcium was also active (0.039 U mg-‘). Figure 5 is data from one experiment with 200 rat lenses. Two preliminary experiments supported these results.
K. J. LAMPI
374
C
E
FIG. 6. Effectof calpastatin and other proteins on calcium-
induced association of calpain with insoluble fraction of rat lens. Lenses from S-week-old rats were homogenized in EGTA buffer bar (A)] or 100 ,UM CaCl, [bar (B)]. Bovine serum albumin [bar (C)l, or trypsin inhibitor [bar (D)], or exogenous calpastatin [bar (E)] were added to lenses homogenized in calcium. Error bars are standard error of the mean for n = 5. * Statistically different from lanes (A) and (E) at P > 0.001. Effect of Calpastatin
The influence of endogenous inhibitor of calpain, calpastatin, upon the calcium-induced insolubilization of calpain was determined (Fig. 6). When no calpastatin was added, 100 ,BM calcium caused a statistically significant increase in cafpain from 0.64 ,ug mg-’ pellet bar (A)] to 1.14 pg mg-’ pellet [bar (B)]. An excess of calpastatin was added in an amount roughly 78 times the estimated calpain activity already present in the test homogenate. With this excess calpastatin, the usual calcium-induced increase in insoluble calpain was prevented [bar (E)]. When BSA roar(C)] or trypsin inhibitor bar (D)] replaced calpastatin with an equal amount of protein (60 pug), calcium still increased calpain binding to the insoluble pellet. This suggested that the inhibitory effect of calpastatin on binding of calpain to the insoluble pellet was specific.
4. Discussion Calpain has been measured in the cytosolic fraction of a wide variety of tissues, including lens (Murachi, 1984). However, the major new findings of the present investigation were: (1) that calpain was associated with several different fractions of the insoluble pellet of lens including a membrane-rich fraction ; (2) calcium induced a reversible transfocation of calpain to the insoluble pellet; and (3) insoluble calpain retained activity which was expressed when the enzyme was resolubilized by washing with chelator. This insoluble calpain is important for several reasons. First, the insoluble pellet may be a site for activation
ET AL.
of calpain. Though calpain is thought to be predominantly cytosolic, its physiological significance is unknown because of the high calcium requirement and presence of cytosolic calpastatin (Murachi. 1984). The membrane was first suggested to play a role in activation of calpain when phospholipids were found to lower its calcium requirement for activation (Pontremoli et al., 1985). This hypothesis was supported when calpain was localized at the membrane in various tissues, and was found to degrade certain membrane-associated proteins (Gopalakrishna and Barsky, 1986). Only 2% of erythrocyte calpain was found at the membrane. Yet, when these membranes were incubated in calcium, band 4.1 was degraded. This suggested a role for calpain in the morphologic change of the erythrocyte membrane (Hatanaka et al., 1984). The association of calpain with the lens membrane may be physiologically significant, because the most prevalent type of calpain in lens is calpain II, requiring mM amounts of calcium for activation. The present study indicated that calpain became associated with the lens insoluble fraction, and this association was increased at calcium concentrations below those required to activate soluble calpain. Furthermore, the membrane may provide a site where calpain may be active in the presence of calpastatin. While the activity ratios of calpastatin to calpain in the lens watersoluble fraction are high, preliminary data from our laboratory showed that this ratio decreased in the insoluble fraction. Also, addition of calcium caused no uptake of calpastatin onto the insoluble pellet. Therefore, calpain may be activated during association with the insoluble fraction, both because of a possible change in its calcium requirement and because of less inhibition from calpastatin. Secondly, binding of calpain to the insoluble pellet is important because it may bring calpain into proximity with its substrates. The lens total insoluble pellet is comprised of different components from the soluble fraction. Calpain may be looseiy associated with the membrane as evidenced by the extraction of waterinsoluble calpain with urea. Urea and alkaline washes strip cytoskeletal and extrinsic proteins from the membrane (Russell, Robinson and Kinoshita, 198 1). Intact rat lenses incubated in calcium resulted in degradation of spectrin and vimentin, implicating calpain in cytoskeletal rearrangement occurring in differentiation of lens fiber cells (Truscott et al., 1990). Calpain may also be an intimate part of the purified membranous fraction, since it was found in the SDSsolubilized pellet that had been urea- and alkalinewashed (Fig. 1, lane 5). The pellet remaining following urea and NaOH washes is a membranous fraction containing mostly 26- and 17-kDa membrane proteins, with some crystallins and other minor proteins (Russell, Robinson and Kinoshita. 1981; Kistler et al., 198 6). Calpain was also in ’ native ’ membrane preparations obtained from a sucrose gradient (Fig. 3).
CALPAIN
AND
INSOLUBLE
PELLET
During cataractogenesis, intracellular calcium increased and specific membrane proteins were degraded, including MP26 (David and Shearer, 1984). In this previous study, calcium concentrations increased to a level high enough to insolubilize calpain, according to the present data. Membranes have been shown to accumulate crystallins during cataract formation (Takeana and Takemoto, 1987), and thus crystallins embedded within the membrane may also be calpain substrates. The demonstrated calciuminduced insolubilization of calpain may allow expression of calpain activity without interference from calpastatin, such as occurs in the water-soluble fraction. Thus, the insoluble pellet might serve to localize substrates for hydrolysis by calpain. The endogenous calpain inhibitor, calpastatin, was also shown to regulate insolubilization of calpain. In rats, the ratio of calpain to calpastatin is about 1: 1 and under such circumstances, calpain was translocated to insoluble pellet when calcium was increased (Fig. 4). However, the translocation of calpain was prevented by great excess of calpastatin over calpain (Fig. 6). This is particularly relevant to the situation in human lens where a considerable excess of calpastatin over calpain exists (David et al., 1989). The rat lens may be a good model for defining the biological function of membranous calpain.
Acknowledgements
The authors wish to thank Dr R. J. Cenedella for expert technical advice. This study was partly supported by NIH grants EY05786 and EYO3600 to T.R.S.
References David, L. L. and Shearer, T. R. (1984). Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest. Ophthalmol. Vis. Sci. 25, 1275-83. David, L. L. andShearer,T. R. (1986). Purification of calpain II from rat lens and determination of endogenous substrates.Exp. Eye Res. 42, 227-38. David, L. L., Varnum, M. D., Lampi,K. J. and Shearer,T. R. (1989). Calpain II in human lens. Invest. Ophthalmol. Vis. Sci. 30, 269-75.
375
Gopalakrishna, R. and Barsky, S. H. (1986). HvdroDhobic associationof calpainswith subcellularorganilles. I. Biol. Chem. 261, 1393642.
Hatanaka, M., Yoshimura, N., Murakami, T., Kannagi, R. and Murachi, T. (1984). Evidence for membraneassociatedcalpain I in human erythrocytes. Detection by an immunoelectrophoreticblotting method using
monospecific antibody. Biochemistry 23, 3272-6. Kistler, J., Kirkland, B., Gilbert, K. and Bullivant. S. (1986). Aging of lens fibers. Invest. Ophthalmol. Vis. Sci. 27. 772-80.
Laemlli, U. K. (19 70). Cleavage of structural proteins during the assemblyof the head of bacteriophageT4. Nature 227, 680-S. Mellgren, R. L., Nettey, M. S.,Mericle, M. T., Renno,W. and
Lane, R. D. (1988). An improved purification procedure for calpastatin, the inhibitor protein specificfor the intracellular calcium-dependentproteinases,calpains. Prep. Biochem. 18, 183-97. Melloni. E., Salamino,F.. Sparatore, B., Michetti. M. and Pontremoli, S. (1984). Ca*+-dependentneutral proteinasefrom human erythrocytes: activation by Ca”+ ions and substrate regulation by the endogenous inhibitor. Biochem. Intern. 8, 477-89. Murachi, T. (1984). Calcium-dependentproteinasesand specific inhibitors: calpain and calpastatin. Biochem. Sot. Symp. 49, 149-67. Pontremoli, S., Melloni, E., Sparatore, B.. Salamino. F., Michetti, M., Sacco.0. andHorecker,B. L. (1985). Role of phospholipids in the activation of the Ca2+-dependent neutral proteinaseof human erythrocytes. Biochem. Biophys. Res. Commun. 129, 389-95.
Russell,P., Robinson,W. G.Jr andKinoshita,J. H. (1981). A new method for rapid isolation of the intrinsic membrane proteinsfrom lens. Exp. Eye Res. 32, 51 l-6. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki,H.. Minami, Y. and Ohno. S. (1988). Regulationof activity of calcium activated neutral protease. Adv. Enzyme Reg. 27. 153-69. Takeana, M. and Takemoto, L. (1987). Quantitation of membrane-associated crystallinsfrom aging and cataractous human lenses.Invest. Ophthalmol. 1%. Sci. 28, 7804. Towbin, H.. Staehelin.T. and Gordan, J. (1979). Electrophoretic transferof proteinsfrom polyacrylamidegelsto nitrocellulosesheets: procedureand someapplications. Proc. Natl. Acad. Sci. U.S.A. 76, 43504.
Truscott, R. J., Marcantonio, J. M.. Tomlinson, J. and Duncan, G. (1990). Calcium-inducedopacificationand proteolysisin the intact rat lens.Invest. Ophthalmol. Vis. Sci. 31, 2405-I 1. Varnum, M. D., David, L. L. and Shearer,T. R. ( 1989). Agerelated changesin calpainII and calpastatinin the rat lens.Exp. Eye Res. 49, 1053-65.
Association KIRSTEN Departments
of Calpain J. LAMPI”,
of Biochemistry
(Received
Norwich
with
LARRY
Insoluble
L. DAVID
AND
Pellet THOMAS
of Rat Lens R. SHEARER
and Ophthalmology, Schools of Dentistry and Medicine, Sciences University, Portland, OR 97201, U.S.A. 78 September
7997 and accepted
in revised form 4 November
Oregon Health
7997)
Calpains are calcium-activated neutral proteases found in many tissues including the lens, The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated Into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of
calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates : and (2) it may activate calpain, since membrane phospholipids lower the protease’s calcium requirement. Key words: calpain ; lens ; calcium ; membrane : calpastatin.
1. Introduction Calpains (CANP, EC 3 .4.22 .17) are cysteine proteases
with neutral pH optima and a requirement for calcium activation (Murachi, 1984). They are found ubiquitously in animal tissues. Calpain II, requiring mM calcium,
is the predominant
form in the lens, and it
has been assayed in several different species. However, the overall concentration of free calcium in normal cells is not high enough to activate calpain II. Data from several laboratories have shown that the calcium activation requirement of calpain was reduced in the or substrates presence of specific phospholipids (Melloni et al., 1984; Pontremoli et al., 1985). It is hypothesized that in the presence of calcium levels below those required for catalytic activity, calpain may associate through an N-terminal hydrophobic domain with phospholipids in the cell membrane (Gopalakrishna and Barsky, 1986; Suzuki et al., 1988). At the membrane, calpain then becomes activated at calcium concentrations below those required for activation of soluble calpain (Suzuki et al., 1988). This may also localize the site of action of calpain and modulate the types of substrates available to it. Nothing is known about the subcellular distribution of calpain in lens fiber cells. The purpose of the present study was to determine if the activation mechanism postulated above may exist in the lens by: (1) measuring
the amount
and activity
of insoluble
* For reprint requests at: Department of Biochemistry, Oregon Health Sciences University, 611 SW. Campus Drive, Portland, OR 97201. U.S.A. 0014-4835/92/080369+07
calpain in the lens; and (2) determining the effect of calcium on the association of calpain with various fractions of the insoluble pellet. We present evidence here that while calpain was predominantly cytosolic in rat lens fiber cells, calpain was also associated with the membrane-rich fraction.
$0%00/O
2. Materials and Methods Animals and Materials
Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Lenses were dissected from enucleated eyes, decapsulated, and the whole lens used for the studies below. Antibody against calpain II was prepared in rabbits by injecting rat muscle calpain II. Calpastatin was purified from bovine heart muscle (Mellgren et al., 1988). Synthetic calpain inhibitors, E64 and a peptide aldehyde CbzValPheH, were obtained from Sigma and as a gift from Merrell-Dow Research Institute (Cincinnati, OH), respectively. isolation of Calpain in Subcelldar Fractions Lenses from 24-day-old rats were homogenized in buffer A containing 20 mM Tris, 1 mM EGTA, 1 mM EDTA, 1 mM DTE, 100 mM KCI, and 2 mM phenylmethylsulfonylfluoride (PMSF), pH 7.5 and centrifuged at 10000 g for 30 min. The insoluble pellet was washed twice in buffer A and then extracted in buffer B, which contained 7 M urea in 50 mM Tris and 1 mM EGTA (pH 7.4) (Russell, Robinson and Kinoshita. 198 1). The urea-insoluble pellet was resuspended in 0.1 M NaOH containing 10 mM /3-mercaptoethanol. 0 1992 Academic Press Limited
K. J. LAMPI
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-
-
Lanes:
I
2
3
4
5
6
7
6
9
(0
1. Immunoblot for calpain in various fractions oflenses from 24-day-old rats. Arrows indicate SO- and 30 kDa subunits of calpain. Lane 1, molecular weight standards; 2, total water-soluble protein: 3. 7 M urea wash of insoluble pellet; 4, 0.1 M NaOH wash of urea-insoluble pellet: 5. remaining insoluble protein. All lanes contained 5Opug protein. Lanes 6-10 contain 2.5, 5, IO, 25, and 50 ng calpain standards. FIG.
Following
centrifugation the remaining insoluble were dissolved in 1% SDS. The above procedure was repeated with 15 9-week-old rats in which one lens from each rat was pooled and treated as above and the other lens processed with 1 mM CaCI, in all the buffers. E64 was included in the initial homogenizing buffer to prevent rapid degradation of calpain in the high calcium buffer. Membrane-rich fractions were obtained using sucrose gradients. Young 13-day-old rat lenses were homogenized in buffer C, which contained 5 mM Tris. 5 mM ,J-mercaptoethanol, 1 mM EGTA, 05 mM PMSF, and 10 ,u~ E64, pH 8.0 (Kistler et al., 1986). The homogenate was split in half and 2 mM CaCl, was added to one of the halves. The insoiuble pellet was isolated by centrifugation at 10000 g for 20 min and washed three times in the same buffer. The washed pellet containing 0.6 mg protein was suspended in 8 % sucrose (by weight) in buffer C and layered into a centrifuge tube loaded with 25, 45, 50 and 55% sucrose solutions for a total volume of 5 ml. The gradient was centrifuged in a Beckman SW 50.1 rotor head (Beckman. Inc. San Francisco, CA) for 90 min at 35 000 rpm. The material at the interfaces was collected, diluted four-fold in buffer C, and recentrifuged for 30 min at 100000 g. The pellets obtained from each interface were resuspended in buffer B and analysed for protein, phosphate, calpain and membrane protein MP26. Phosphate and MP26 were used as membrane markers. Protein was measured using proteins
ET AL.
the BioRad protein assay (BioRad. Richmond. CA). Phosphate content was determined with FiskeSubbarow reagent according to manufacturer’s directions (Sigma. St Louis, MO) and involved an organic extraction in chloroform : methanol ( 1: 1) followed by perchloric acid digestion. MP26 was identified from Coomassie blue stains of SDS-PAGE gels. Calpain was identified by immunoblotting as described below. Measurement of Calcium Dependency and Reversibility of Calpain Insolubilization The calcium dependency for binding of calpain to insoluble pellet was determined by homogenizing lenses from S-week-old rats in the presence of increasing amounts of added calcium (0 to 1 mM). The calpain inhibitor peptide CbzValPheH was added at 0.24 mM to prevent autolysis and calpain was quantitated by immunoblotting. CbzValPheH was chosen because it was more readily available in our laboratory than E64 at the time this experiment was conducted. Calpain activity and immunoreactivity were measured in water-soluble, chelator-extractable, and Triton X-100 solubilized proteins from 200 rat Ienses. age 24 days. Homogenization was carried out in 40 ml of 20 mM Tris (pH 7.51, 100 ,u~ EGTA, 2 mM DTE and 0.5 mM PMSF at 4°C. The homogenate was split in half and 200 /LM CaCl, was added to one portion. This represents both the EGTA-bound and -free calcium. The pellets were washed three times in the appropriate buffer. Calpain was extracted from the total insoluble pellets by homogenization in buffer A. without 100 mM KC1 three times. This calpain was designated ‘chelator-extract,able ‘. The remaining pellets were extracted overnight in 1% Triton X-100 followed by sonication. Triton was then removed using Pierce Extracti-gel (Pierce, Rockford, IL). All samples were applied to a TSK DEAE S-PW column and proteins eluted with a linear gradient of 0 to 0.5 M NaCl in buffer A. Calpain activity was assayed using FITClabeled casein (David and Shearer, 1986). One unit of calpain activity equaled 1 fig FITC-labeled, acidsoluble fragments released per min. Enzyme-linked immunosorbent assays @LISA) on DEAE fractions were formed using calpain II antibody. Electrophoresis and lmmunoblotting SDS-PAGE of various subcellular fractions was performed using 12 % acrylamide gels (Laemlli, 19 70). Proteins were then electrophoretically transferred to PVDF membrane (Millipore. Bedford, MA) using the Towbin buffer system (Towbin, Staehelin and Gordan, 19 79). Blots were incubated with anticalpain antibody and developed by alkaline phosphatase as recommended by the manufacturer (BioRad, Richmond, CA). Intensity was estimated by densitometry using a Hoefer GS-300 densitometer (Hoefer Scientific Instruments, San Francisco, CA). The amount of calpain in
CALPAIN
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371
TABLE I Effect of calcium on association of calpain with various lens fractions from g-week-old Homogenization in EGTA /lg calpain per mg protein
Subcellular fraction
CaCI,
% of total calpain
fig calpain per mg protein
% of total calpain
90.5 3.2 5.4 0.9 9.5
042 + 0.06* 2.09kO.22 0.97kO.09 1~90+0~10
61.7 19.6 15.9 2.8 38.3
1.00 +0.05* 0.59kO.07 0.42 + 0.10 0.93f0.20
Water-soluble Urea-soluble NaOH-soluble Pellet Total insoluble
Homogenization in 1 rn~
rats
* Meanks.D. 01= 5).
EGTA
Calcium
I
97
I
1
I
kDo
66
45
31
-
,MP
26
22
14
Lanes:
I
2
3
4
5
6
7
a
9
IO
FIG. 2. SDS-PAGE gel of 13-day-old rat lens insoluble pellet stained with Coomassie blue showing MP26 at the interfaces from a discontinuous sucrose gradient. The same volume was applied to each lane from identically treated samples. Lanes 1, 2, 3 and 4 are the g/25%, 25/45%, 45/50% and 50/55% sucrose interfaces, respectively. Lanes 6, 7, 8 and 9 are the same interfaces from a sucrose gradient in which 2 mM calcium was added to all the buffers, including the initial homogenate. Lanes 5 and 10 are from the pellets of the above gradients. unknown samples was calculated from a standard curve produced by adding known amounts of rat muscle calpain to the same immunoblot containing the unknown samples.
3. Results Distribution
of Calpain in Various Fractions of Lens
Calpain was found in all lens fractions isolated (Fig. 1). An immunoreactive band corresponding to the
80-kDa subunit of calpain was found in the watersoluble (cytosolic), urea-soluble (calpain easily dissociated from pellet), NaOH-soluble (calpain tightly bound to pellet), and SDS-soluble fractions (calpain possibly imbedded within membrane). Based on the densitometric measurements of the 80-kDa band, the majority of calpain, 94.2% was located in the cytosolic fraction when lenses were homogenized in the absence of calcium (Fig. 1). Significant portions of lens calpain were also loosely and tightly bound to the insoluble pellet. Relative amounts of 5.0%, 07 % 24-2
K. J.
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ET AL.
TABLE II
and 0.1 y0 of total calpain were found in the urea-, NaOH- and SDS-extracted fractions (Fig. 1). The
Distribution of phosphatesin discontinuous sucrose
total protein isolated from the same four fractions was 88.8 mg of water-soluble protein, 6.5 mg of ureasoluble protein, 1.3 mg of NaOH-extracted protein, and 0.4 mg protein remaining in the insoluble pellet. The percentage of protein obtained in each fraction was similar to the percentages of calpain in each fraction. When lenses were homogenized in 1 mM CaCl,, calpain was redistributed from the cytosol into all fractions of the insoluble pellet (Table I). Individual insoluble fractions showed calcium-induced increases in calpain ranging from three- to six-fold. Insoluble calpain increased from 9.5% of the total lens calpain to 38.3% due to the addition of calcium, with the greatest increase in the urea-soluble fraction. These increases in the insoluble fraction were accompanied by a 29% decrease in the amount of total watersoluble calpain. The loss of soluble calpain was due to translocation of calpain to the insoluble pellet in the presence of calcium. This loss of soluble calpain was not due to a generalized insolubilization of lens proteins. The amount of insoluble protein did not increase. There was 6.2 mg of total insoluble protein from lenses homogenized in EGTA buffer and 56 mg of protein from lenses homogenized in calcium buffer. Insoluble calpain, however, increased from 3.1 to 7.9 ,ug. This indicated that the calcium-induced effect was specific for calpain. A greater percentage of insoluble calpain was present in 9-week-old lenses than in 24-day-old lenses (9.5 compared with 5*8x, respectively). As expected,
gradient
Interface
Homogenizationin EGTA nmol phosphates per j/g protein
Homogenizationin 1 mM CaCl, nmol phosphates per jig protein
S/25% 25145% 45/50X 50/55% Pellet
0.0 0.17 0.72 0.72 0.1 I
04 O,h7 0.76 (bl 3 0.2 3
total insoluble lens protein was larger in older rats than in younger ones (17.4 compared with 8.4x, respectively). Insoluble calpain and insoluble protein in the lens have been documented to increase with age (Takeana and Takemoto, 198 7 : Varnum, David and Shearer, 1989). The mechanism for the age-related calcium-induced insolubilization of calpain was not examined in this study. To obtain a more native membrane preparation, without the use of urea or NaOH, the insoluble pellet was separated on a discontinuous sucrose gradient. Material at the 25/45X, 45150% and 50155% interfaces was found to contain MP26 as the major protein (Fig. 2, lanes 2, 3,4). Phosphates, a marker for phospholipids, were also found at these interfaces (Table II). Some MP26 and phosphates pelleted at the bottom of the gradient (Fig. 2, lane 5; Table II).
EGTA
Calcium
Standards I
97
LAMPI
I
I
kDo
t
60 kDO
66
43
31
I
2
3
4
5
6
?
6
9
IO
II
12
I3
14
FIG.3. Immunoblot for calpainin 13-day-oldrat lensinsolublepelletfrom the interfacesof a discontinuoussucrosegradient. Seelegendto Fig. 2 for descriptionof lanes l-10. Lanes11, 12, 13. and 14 are 2.5, 5. 15 and 30 ng of calpain standards. respectively.
CALPAIN
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373
fractions as marked by MP26 and phosphates, and calcium increased this association. At the same time that calcium increased calpain association with the membrane, an absence of proteins above 3 1 kDa was observed (Fig. 2, lanes 7-9), and a decrease and shifting of bands was noted (Fig. 2, lane 10). This indicated that the concentration of E64 in these studies was not high enough completely to inhibit proteolysis by calpain or that other calcium proteases may exist in the lens. Calcium also altered the distribution of phosphates. The significance of this is unclear. Calcium Dependencyof the Association of Calpain with the InsoIubIePellet pt.4 calcium
FIG. 4. Calcium dependency curve for binding of calpain to lens insolnble pellet from 5-week-old rats. Each dot is an average of four determinations, with an average standard error of the mean = + 0.18 ,ug mg-I. Amount of calpain in pellet at 500 ,LLM calcium was greater than with 0 added calcium (P > 0.05).
3oI
To determine the calcium dependency for binding of calpain to the insoluble fraction, lenses from S-weekold rats were homogenized in buffers containing increasing amounts of calcium. With increasing calcium, more calpain became associated with the insoluble pellet (Fig. 4). The most dramatic increase in the insoluble calpain was seen when added calcium was elevated from 0 to 50 PM. The amount of calpain associating with pellet started to plateau at approximately 500 ,UM and a final three-fold increase over starting levels was observed at 1000 ,BM calcium. Reversibility of Calpainlnsolubilization
Calpain association with the insoluble pellet due to calcium was reversible, and the resolubilized enzyme remained active. Lenses were homogenized with and without 200 PM-free CaCl,, and the resulting insoluble pellets washed with high-chelator buffer. Lenses were homogenized at a calcium level chosen to increase insoluble calpain without causing extensive autolysis
FIG. 5. Specific amount of calpain in chelator [lane (B)] and Triton X-100 [lane (C)] washes of the insoluble pellet compared to water-soluble calpain [lane (A)]. q , Control values. q , Values from lenses homogenized in 200,uM
CaCl,. Lenses were from 24-day-old rats. Immunoblots showed calpain to be present in the same interfaces where MP26 was observed (Fig. 3, lanes 2, 3, 4). Calpain was not present at the 8/25X interface (Fig. 3, lane 1) and a dark-staining band of calpain was located at the bottom of the 55 % layer (Fig. 3, lane 5). This latter dense calpain was apparently not associated with lipid component. Adding calcium at a final concentration of 1 mM to the homogenizing buffer increased calpain found at the 25/45x, 45/50X and !iO/SS% interfaces (Fig. 3, lanes 7, 8, 9). Calcium did not alter the amount of MP26 found in these fractions. Calcium also increased the amount of calpain which pelleted (Fig. 3, lane 10). Thus. calpain was associatedwith the membrane-rich
and degradation of calpain. Four times more calpain was extracted from the pellet isolated in the presence of calcium than from the control pellet (Fig. 5). Calpain bound to the pellet due to calcium was resolubilized with chelator washes. The specific activity of this chelator-extractable calpain was 0.153 U mg-‘. This was greater than the water-soluble calpain activity (0.011 U mg-‘). The measurable activity was due to calpain because it eluted off a DEAE column at a salt concentration characteristic of calpain (David and Shearer, 1986). and it was completely inhibited by 100 ,UM E64, a synthetic cysteine protease inhibitor. The active fractions were also immunoreactive with calpain antibody in an ELISA assay. Some of the insolubilized calpain remained insoluble after several chelator washes and was only removed by Triton (Fig. 5). The activity of this Triton-solubilized calpain was 0105 U mg-‘. Calpain extracted with Triton from the insoluble pellets of lenses homogenized without added calcium was also active (0.039 U mg-‘). Figure 5 is data from one experiment with 200 rat lenses. Two preliminary experiments supported these results.
K. J. LAMPI
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C
E
FIG. 6. Effectof calpastatin and other proteins on calcium-
induced association of calpain with insoluble fraction of rat lens. Lenses from S-week-old rats were homogenized in EGTA buffer bar (A)] or 100 ,UM CaCl, [bar (B)]. Bovine serum albumin [bar (C)l, or trypsin inhibitor [bar (D)], or exogenous calpastatin [bar (E)] were added to lenses homogenized in calcium. Error bars are standard error of the mean for n = 5. * Statistically different from lanes (A) and (E) at P > 0.001. Effect of Calpastatin
The influence of endogenous inhibitor of calpain, calpastatin, upon the calcium-induced insolubilization of calpain was determined (Fig. 6). When no calpastatin was added, 100 ,BM calcium caused a statistically significant increase in cafpain from 0.64 ,ug mg-’ pellet bar (A)] to 1.14 pg mg-’ pellet [bar (B)]. An excess of calpastatin was added in an amount roughly 78 times the estimated calpain activity already present in the test homogenate. With this excess calpastatin, the usual calcium-induced increase in insoluble calpain was prevented [bar (E)]. When BSA roar(C)] or trypsin inhibitor bar (D)] replaced calpastatin with an equal amount of protein (60 pug), calcium still increased calpain binding to the insoluble pellet. This suggested that the inhibitory effect of calpastatin on binding of calpain to the insoluble pellet was specific.
4. Discussion Calpain has been measured in the cytosolic fraction of a wide variety of tissues, including lens (Murachi, 1984). However, the major new findings of the present investigation were: (1) that calpain was associated with several different fractions of the insoluble pellet of lens including a membrane-rich fraction ; (2) calcium induced a reversible transfocation of calpain to the insoluble pellet; and (3) insoluble calpain retained activity which was expressed when the enzyme was resolubilized by washing with chelator. This insoluble calpain is important for several reasons. First, the insoluble pellet may be a site for activation
ET AL.
of calpain. Though calpain is thought to be predominantly cytosolic, its physiological significance is unknown because of the high calcium requirement and presence of cytosolic calpastatin (Murachi. 1984). The membrane was first suggested to play a role in activation of calpain when phospholipids were found to lower its calcium requirement for activation (Pontremoli et al., 1985). This hypothesis was supported when calpain was localized at the membrane in various tissues, and was found to degrade certain membrane-associated proteins (Gopalakrishna and Barsky, 1986). Only 2% of erythrocyte calpain was found at the membrane. Yet, when these membranes were incubated in calcium, band 4.1 was degraded. This suggested a role for calpain in the morphologic change of the erythrocyte membrane (Hatanaka et al., 1984). The association of calpain with the lens membrane may be physiologically significant, because the most prevalent type of calpain in lens is calpain II, requiring mM amounts of calcium for activation. The present study indicated that calpain became associated with the lens insoluble fraction, and this association was increased at calcium concentrations below those required to activate soluble calpain. Furthermore, the membrane may provide a site where calpain may be active in the presence of calpastatin. While the activity ratios of calpastatin to calpain in the lens watersoluble fraction are high, preliminary data from our laboratory showed that this ratio decreased in the insoluble fraction. Also, addition of calcium caused no uptake of calpastatin onto the insoluble pellet. Therefore, calpain may be activated during association with the insoluble fraction, both because of a possible change in its calcium requirement and because of less inhibition from calpastatin. Secondly, binding of calpain to the insoluble pellet is important because it may bring calpain into proximity with its substrates. The lens total insoluble pellet is comprised of different components from the soluble fraction. Calpain may be looseiy associated with the membrane as evidenced by the extraction of waterinsoluble calpain with urea. Urea and alkaline washes strip cytoskeletal and extrinsic proteins from the membrane (Russell, Robinson and Kinoshita, 198 1). Intact rat lenses incubated in calcium resulted in degradation of spectrin and vimentin, implicating calpain in cytoskeletal rearrangement occurring in differentiation of lens fiber cells (Truscott et al., 1990). Calpain may also be an intimate part of the purified membranous fraction, since it was found in the SDSsolubilized pellet that had been urea- and alkalinewashed (Fig. 1, lane 5). The pellet remaining following urea and NaOH washes is a membranous fraction containing mostly 26- and 17-kDa membrane proteins, with some crystallins and other minor proteins (Russell, Robinson and Kinoshita. 1981; Kistler et al., 198 6). Calpain was also in ’ native ’ membrane preparations obtained from a sucrose gradient (Fig. 3).
CALPAIN
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PELLET
During cataractogenesis, intracellular calcium increased and specific membrane proteins were degraded, including MP26 (David and Shearer, 1984). In this previous study, calcium concentrations increased to a level high enough to insolubilize calpain, according to the present data. Membranes have been shown to accumulate crystallins during cataract formation (Takeana and Takemoto, 1987), and thus crystallins embedded within the membrane may also be calpain substrates. The demonstrated calciuminduced insolubilization of calpain may allow expression of calpain activity without interference from calpastatin, such as occurs in the water-soluble fraction. Thus, the insoluble pellet might serve to localize substrates for hydrolysis by calpain. The endogenous calpain inhibitor, calpastatin, was also shown to regulate insolubilization of calpain. In rats, the ratio of calpain to calpastatin is about 1: 1 and under such circumstances, calpain was translocated to insoluble pellet when calcium was increased (Fig. 4). However, the translocation of calpain was prevented by great excess of calpastatin over calpain (Fig. 6). This is particularly relevant to the situation in human lens where a considerable excess of calpastatin over calpain exists (David et al., 1989). The rat lens may be a good model for defining the biological function of membranous calpain.
Acknowledgements
The authors wish to thank Dr R. J. Cenedella for expert technical advice. This study was partly supported by NIH grants EY05786 and EYO3600 to T.R.S.
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Gopalakrishna, R. and Barsky, S. H. (1986). HvdroDhobic associationof calpainswith subcellularorganilles. I. Biol. Chem. 261, 1393642.
Hatanaka, M., Yoshimura, N., Murakami, T., Kannagi, R. and Murachi, T. (1984). Evidence for membraneassociatedcalpain I in human erythrocytes. Detection by an immunoelectrophoreticblotting method using
monospecific antibody. Biochemistry 23, 3272-6. Kistler, J., Kirkland, B., Gilbert, K. and Bullivant. S. (1986). Aging of lens fibers. Invest. Ophthalmol. Vis. Sci. 27. 772-80.
Laemlli, U. K. (19 70). Cleavage of structural proteins during the assemblyof the head of bacteriophageT4. Nature 227, 680-S. Mellgren, R. L., Nettey, M. S.,Mericle, M. T., Renno,W. and
Lane, R. D. (1988). An improved purification procedure for calpastatin, the inhibitor protein specificfor the intracellular calcium-dependentproteinases,calpains. Prep. Biochem. 18, 183-97. Melloni. E., Salamino,F.. Sparatore, B., Michetti. M. and Pontremoli, S. (1984). Ca*+-dependentneutral proteinasefrom human erythrocytes: activation by Ca”+ ions and substrate regulation by the endogenous inhibitor. Biochem. Intern. 8, 477-89. Murachi, T. (1984). Calcium-dependentproteinasesand specific inhibitors: calpain and calpastatin. Biochem. Sot. Symp. 49, 149-67. Pontremoli, S., Melloni, E., Sparatore, B.. Salamino. F., Michetti, M., Sacco.0. andHorecker,B. L. (1985). Role of phospholipids in the activation of the Ca2+-dependent neutral proteinaseof human erythrocytes. Biochem. Biophys. Res. Commun. 129, 389-95.
Russell,P., Robinson,W. G.Jr andKinoshita,J. H. (1981). A new method for rapid isolation of the intrinsic membrane proteinsfrom lens. Exp. Eye Res. 32, 51 l-6. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki,H.. Minami, Y. and Ohno. S. (1988). Regulationof activity of calcium activated neutral protease. Adv. Enzyme Reg. 27. 153-69. Takeana, M. and Takemoto, L. (1987). Quantitation of membrane-associated crystallinsfrom aging and cataractous human lenses.Invest. Ophthalmol. 1%. Sci. 28, 7804. Towbin, H.. Staehelin.T. and Gordan, J. (1979). Electrophoretic transferof proteinsfrom polyacrylamidegelsto nitrocellulosesheets: procedureand someapplications. Proc. Natl. Acad. Sci. U.S.A. 76, 43504.
Truscott, R. J., Marcantonio, J. M.. Tomlinson, J. and Duncan, G. (1990). Calcium-inducedopacificationand proteolysisin the intact rat lens.Invest. Ophthalmol. Vis. Sci. 31, 2405-I 1. Varnum, M. D., David, L. L. and Shearer,T. R. ( 1989). Agerelated changesin calpainII and calpastatinin the rat lens.Exp. Eye Res. 49, 1053-65.