4RCHIVES
OF
BIOCHEMISTRY
Trypsin
AND
Digestion
BIOPHYSICS
126,469-477
of Fragmented G. INESP
University
of the Pacijk
and
University
AND
(1968)
Sarcoplasmic
Reticulum’
H. ASAI
of California3
San Francisco,
Received December 7, 1967; accepted February
California
9@%?
6, 1968
Short incubation of fragmented sarcoplasmic reticulum with trypsin produces inhibition of ATP dependent calcium accumulation, while ATPase is activated. The addition of trypsin to loaded vesicles causes a rapid calcium leak, suggesting that certain easily digestible peptidic bonds may contribute to the maintenance of semipermeability in sarcoplasmic membranes. A small amount of calcium is still taken up by partially digested membranes, independently of the presence of ATP. This uptake is due to binding to the membrane itself and is competitively inhibited by other cations. A’I!Pase is inhibited by longer digestions, while obvious structural changes appear throughout the thickness of the membrane. This indicates that either ATPase is extended into the deeper layer of the membrane, or it is very resistant to trypsin attack. A parallel pattern of inhibition is produced by trypsin on ATPaae and ADP-ATP exchange, indirectly suggesting interdependence of these two activities. Depending on the time of digestion, more or less marked alterations of the membrane structure can be observed on electronmicroscopy (negative staining with phosphotungstic acid) : disappearance of an outer granular layer, irregular thickness of the membrane, opening of the vesicles and aggregation. Suspensions of fragmented sarcoplasmic reticulum digested with trypsin, undergo a rapid decrease in turbidity, likely due t.o a change in the state of s.ggregation nf the vesicles.
Vesicular fragments of sarcoplasmic reticulum have been isolated from skeletal muscle and shown to be able to produce relaxation of contractile models, accumulate calcium and catalyze Ca dependent ATP hydrolysis and ADP-ATP exchange (1, 2, 3, 4, 5). These activities of the fragmented sarcoplasmic reticulum can be modified by several denaturing agents (6). Of particular interest are the results reported by Martonosi (6, 7), who obtained inactivation of ATPase and calcium uptake following treatment with phospholipase C. On the 1 Supported by USPHS HE-09878 and AHA 66-742. * Established Investigator of the American Heart Association. 3 Reprint requesm to Dr. G. Inesi, Cardiovascular Research Institute, University of California, San Francisco Medical Center, San Francisco, California 94122. 469
other hand, the obvious importance of the membrane protein component was indicated by Nagai et al. (S), who found that trypsin treated sarcoplasmic granules were not able to inhibit myofibrillar ATPase. The experiments described in this report were designed to study the effect of stepwise trypsin digestion of fragmented sarcoplasmic reticulum, following the modifications of calcium uptake, ATP hydrolysis and ADPATP exchange, and observing the changes induced on the membrane structure. METHODS Fragmented sarcoplasmic reticulum was obtained from rabbit skeletal muscle by homogenization and differential centrifugation, as previously described (9, 10). ATP dependent calcium uptake was determined by measuring the residual 45Ca after removal of the fragmented membranes by Millipore (HA 0.45 p) filtration. The reaction mixture con-
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tained: 20 rn~ Tris-Maleate (pH 6.8), 80 mM KCl, 5 mM MgClz, 4-5 mivr ATP, 0.1 mM EGTA,’ 0.1 mM CaClz and variable concentrations of membrane protein. ATP hydrolysis was followed by measuring inorganic phosphate by the molybdo-vanadate method (11, 12). The reaction mixture was identical to that used for calcium uptake. ADP-ATP exchange was st,udied determining the conversion of 1% uniformly labeled ADP to ATP. The reaction mixture was identical to that used for calcium uptake, except for the fact that it contained 2 mM 1% ADP-ADP and 2 mM ATP, instead of 5 mM ATP. The reaction was stopped by removing the membrane fragments by Millipore filtration. 5 ml aliquots of the filtrate were cooled in ice, diluted to 50 ml with cold water and passed through a column containing 3 ml of anion exchange resin (AG l-X8 Bio-Rad, lo&200 mesh). Fractional elution of AMP, ADP, and ATP was then obtained with 0.005 N, 0.02 N and 0.1 N HCI. The entire chromatographic procedure was carried out at 2-3”. The adenosine moiety concentration in the samples was calculated from the uv absorption (h 260), and the radioactivity measured by scintillation spectrometry. The effect of trypsin on the vesicles was studied either by adding trypsin5 directly to a reaction mixture while an experimental parameter was being measured (calcium uptake, optical density) or by incubating the membranes with trypsin before the experiments were performed. Preincubations were started by adding trypsin (1 mg/ 100 mg membrane protein) to several test tubes containing 80 mM KCI, 20 mM Tris-Maleate buffer (pH 6.8) and approximately 2.5 mg membrane protein per ml. The digestion was stopped at different times by adding 2 mg of trypsin inhibitor5 per 1 mg of trypsin. All the samples and the control (to which inactivated trypsin was added) were kept at 25” for the same time. Aft,er centrifugation at 40,OOOg for 2 hours, the pellets were resuspended in 10 InM Tris-Maleate buffer (pH 6.8). Droplets of membrane Electronmicroscopy. containing approximately .l mg suspensions, membrane protein per ml, were placed on copper grids covered with a carbon-collodium film. After 2-3 minutes, the excess water was removed by touching the edge of the grid with filter paper. A drop of 1% phosphotungstic acid (brought to pH 5.8 with KOH) was applied for several seconds and the excess removed with filter paper. The * Ethylenglycol-bis (p-aminoethylether)-N,N’tetraacetic acid. 5 Trypsin and soybean trypsin inhibitor purchased from Sigma Chemical Company.
were
AND
ASAI preparations were t,hen allowed to dry and examined on a Philips EM 200 with a double condenser illumination (300 ~1 platinum apertures), a 40 fi plat.inum objective aperture and accelerating voltage of 60 kv. Most of the pictures were taken at magnification of 46,400 using electron image plates purchased from Eastman Kodak co. RESULTS
TURBIDITY
CHANGES
A readily apparent change in a suspension of fragmented sarcoplasmic reticulum undergoing trypsin digestion, is a reduction in turbidity. This can be easily followed by adding, directly in the cuvette of a spectrophotometer, an appropriate amount of trypsin to a suspension of membrane fragments. The change begins rapidly, but proceeds at a gradually reduced rate (Fig. 1). No significant turbidity change is produced by the addition of a mixture of trypsin and trypsin inhibitor. The values plotted in Fig. 1 were obtained using a 450 rnp wavelength. It should be pointed out that two parallel logarithmic plots can be obtained as the result of wavelength scanning (350-800 mK) before and after 60 minutes of digestion (digestion interruped by the addition of trypsin inhibitor). RELEASE OF PROTEIN suspensions were When membrane digested with trypsin and then centrifuged at 40,OOOg for 2 hours, the amount of protein recovered in the sediment was decreased, as compared to controls incubated with inactivated trypsin. An amount of protein sufficient to match the difference could be determined in the supernatant. The plot of residual protein versus time of digestion (Fig. 2), shows that the release of protein from the vesicles occurs at a faster rate during the first 15 minutes of digestion, and at a slower rate thereafter. CALCIUM UPTAKE The time course of net calcium uptake by fragmented sarcoplasmic reticulum is shown in Graph No. 3, where the “maximal filling capacity” (in the absence of oxalate) is
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FIG. 1. Optical density (450 rnp) of fragmented sarcoplasmic reticulum suspended in 20 rnM Tris-Maleate (pH 6.8) and 80 mu KCl. The suspension contained .69 mg membrane protein/ml. At time 0, trypsin or trypsin + trypsin inhibitor (1 and 2 mg yG mg membrane protein) were added to the cuvette of a spectrophotometer. No sedimentation occurred during the time of reaction, and stirring of the suspension did not alter optical changes per
reached in approximately 2 minutes. The addition of inactivated trypsin does not alter this equilibrium, as compared to controls with no addition. On the contrary, active trypsin produces a rapid loss of calcium from the loaded vesicles. Experiments on calcium uptake by membranes predigested for different lengths of time, centrifuged and resuspended, demonstrated that the ATP dependent net uptake is completel,y lost in vesicles predigested for ten minutes with 1 mg trypsin per 100 mg membrane protein (Fig. 4). The low capacity for calcium binding, which is displayed by the membrane even in the absence of ATP, is more resistant and is slowly reduced by longer incubations with trypsin. In a semilogarithmic plot (Fig. 5) of calcium uptake as a function of time of digestion, it can be easily recognized that
the ATP dependent net uptake showsa very steep slope of inhibition. The remaining net uptake, which is inhibited more gradually by longer digestions, does not require ATP and is even greater in the absence of both ATP and Mg. One should recall that, as outlined in the section on methods, the trypsin digestion was carried out by incubating for the same length of time several tubes containing suspensions of membrane fragments with trypsin. The digestion was stopped by the addition of trypsin inhibitor at different times. In the controls, a mixture of t,rypsin and inhibitor was added at the beginning of the incubation. Controls incubated with inactivated trypsin, or bovine albumin (albumin : membrane protein = 3 : loo), or no addition, retained the same capacity for calcium accumulation.
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FIG. 2. Protein loss by fragmented sarcoplasmic reticulum digested with trypsin. React.ion mixture: 20 mM Tris-Maleate (pH 6.8), SO mM KCI, 1.7-1.9 mg membrane protein/ml. Several samples were incubated with trypsin (I mg ‘$$ mg membrane protein) at 25”. The reactions were stopped at different times by adding trypsin inhibitor (2mg/l mg trypsin). A mixture of trypsin and trypsin inhibitor was added to the control. All the samples were kept at 25” for the same time. Centrifugation at 40,OOOg (2’) was then carried out for 2 hours and the protein in the supernatant and in the resuspended sediment was determined. The values reported on this graph indicate the residual protein in the sediment of samples digested for different times, as compared to a cont,rol incubated with inactivat,ed trypsin.
ASAI
ATPase: slight activation by a short digestion, and inactivation by longer digestions. A semilogarithmic plot of activity versus time of digestion shows a similar slope of inhibition for both ATPase and ADP-ATP exchange (Fig. 5). The figures in the graphs illustrating changesin ATPase and ADP-ATP exchange are expressed as activity per mg of original protein (the amount of protein found in membranes centrifuged after incubation with inactivated trypsin). If the activity is expressed per mg of residual protein (the amount of protein found in each of the membrane samples centrifuged after different times of trypsin digestion) higher values and a slower inactivation are obtained. In fact, calculating from the figures reported on Graph No. 2 and 6, it can be easily seenthat
16Or
Trypsin
t
inhibitor
ATP HYDROLYSIS
AND ADP-ATP EXCHANGE
While calcium accumulation is reduced, ATPase activity is increased in membranes incubated for a short time with trypsin. This is best shown in vesicles predigested for 10 minutes (Fig. 5). Longer preincubations with trypsin produce inactivation of ATPase. It should be poimed out that the residual ATPase of partially digested membranes is still calcium dependent; in fact, very little activity is obtained when no calcium is added to a reaction mixture containing EGTA (Fig. 6). ADP-ATP exchange activity is modified hv trvnsin with a nattern similar to that of
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FIG. 3. Calcium release by loaded vesicles on addition of trypsin. Reaction mixture: 20 mM Tris-Maleate buffer (pH 6.8), 80 mM KCl, 5 mM MgCl*, 5 rnM ATP, .I mM EGTA, .l mM CaCh, .38 membrane protein/ml. The reaction (25”) was started by the addition of ATP. After 1 minute, trypsin or trypsin + trypsin inhibitor (1 and 2 mg y0 mg membrane protein) were added to the reaction mixture.
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. ATP and Mg added 0 Mg added No ATP added ANeither M g or ATP added
Calcium upbake ,ATP dependent,
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FIG. 4. Net calcium uptake by fragmented sarcoplasmic reticulum predigested with trypsin (see section on methods and legend of Fig. 2). Reaction mixture: 20 mM Tris-Maleate (pH 6.8), 80 rnM KCl, .l mM EGTA, .I mM CaC12, .481.19 mg membrane protein/ml; 5 mM MgClz and 5 rnM ATP (when indicated). Incubation for 2 minutes at 25”. after 180 minutes of trypsin digestion, the specific activity is .9 ctmoles/min/mg residual protein for ATPase, and 1.96 ~moles/min/mg residual protein for ADPATP exchange. Neither ATPase activity nor ADP-ATP exchange were catalyzed to a significant extent by the protein recovered from the supernatant, after centrifugation of membranes digested with trypsin. MEMBRANE STRUCTURE An example of the appearance of fragmented sarcoplasmic reticulum, negatively stained with phosphotungstic acid, is reported in plate 1. The vesicles, of a diameter varying between .125 and .25 p, are of a rounder shape than those observed on thin sections of fixed material (l-lo). The difference is probably due to osmot.ic effects of staining reagents or interaction of the membrane with the carbon film on the grid. An outer layer of repeating particles can be clearly seen on the negatively stained membranes (Fig. 7). The shape of these particles is not ~$1 outlined owing to their small size (30-40 A).
(ATP independent]
s
FIG. 5. Semilogarithmic plot of net calcium uptake (in the presence: A and in the absence of ATP: A), ATPase and ADP-ATP exchange activities, as a function of time of preincubation of the membrane fragments with trypsin. Experimental conditions for calcium uptake as described in the legend for Fig. 4. For ATP hydrolysis and ADP-ATP exchange: 20 mM Tris-Maleate (pH 6.8), 80 mM KCl, 4 mM MgC12, 4 mM ATP or 2 mM ADP + 2 mM ATP (exchange reaction), 36-84 rg membrane protein/ml, .l mM EGTA, .I mM CaCl,; incubations carried out at 25” and 4 serial samples taken at I-4-minute intervals; results expressed as rates derived from linear activities. Trypsin, depending on the time of incubation, produces more or less marked changes of the membrane structure. The outer granular layer disappears after 10-20 minutes of digestion. The digested fragments tend to aggregate while control vesicles always maintain a space between each other. As the digestion proceeds, the thickness of the membrane becomes irregular and several vesicles show discontinuity of the limiting membrane. Further digestion produces fragmentation of the vesicles. A representation of the changes above described is shown in Fig. 8.
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ANI)
ARAI
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FIG. 6. ATP hydrolysis and ADP-ATP exchange by fragmented sarcoplasmic predigestSed with trypsin (see section on methods and legend of Fig. 2). Reaction as described in leg&d of Fig. 5.
reticulum mixture:
albumin, or no addition, retain the same The inhibition of calcium accumulation capacity for calcium accumulation. A high residual ATPase activity is found and enhancement of ATP hydrolysis proin partially digested sarcoplasmic memduced by short incubation with trypsin (Fig. 5) is quite similar to that obt#ainedby branes. A significant inhibition can only be treating fragmented sarcoplasmic reticulum produced by long incubations with trypsin with small volumes of diethyl ether (10). (Fig. 5), when obvious changes within the These effects appear to be due to increased thickness of the membrane are observed on membrane permeability and calcium leak, electronmicroscopy. This finding is in agreement with a recent report by Marchesi as it is shown that loaded vesicles rapidIy lose calcium after addition of trypsin to the and Palade (13), who showed that inactivareaction mixture (Fig. 3). In the treated tion of ATPase and structural breakdown vesicles, calcium dependent ATP hydrolysis are produced simultaneously by t,rypsin is maintained at high constant rate due to digestion of red cell membranes. This may the fact that the calcium concentrations indicate that the enzyme is extended into outside and inside the vesicles remain the deeper layer of the membrane. Our experiments are similar to those of Oda and constant. The finding that such an alteration is produced by trypsin digestion indicates Seki (14), who found that while outer that, in sarcoplasmic reticulum, certain particle can be removed by papain digestion easily digestible peptide bonds may con- of isolated intestinal microvilli, ATPase tribute to the maintenance of membrane activity remains associated with the basal semipermeability. The possibility should be membrane layer. In addition, these authors considered that nonspecific interaction of produced histochemical evidence indicating trypsin with the vesicles could also interfere that ATPase activity is JocaIized predomiwith the semipermeability properties of the nantly in the inner leaflet. of the trilaminar membrane structure. On the other hand, membrane. However, the ratio of trypsin: have reported membrane protein used in our experiments Hasselbach and Elfvin (l:lOO), and the time of incubation neces- evidence indicating that certain SH-groups, sary for the development’ of full effect, specifically involved in ATPase activity, minimize the role of this factor. Controls are located on the outer surface of sarcoincubated with inactivated trypsin, or plasmic vesicles (15). If all the ATPase is
DIGESTION
exclusively layer, our postulating to trypsin structural
OF
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located on the outer membrane results should be explained by that the enzyme is more resistant attack, as compared to other proteins.
FIG. i’. Fragmented acid. X232,000.
sarcoplasmic
ret,iculum,
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RETICIJLIJM
The modification of ADP-ATP exchange act’ivity induced by trypsin follows a pattern very similar to that of ATPase modification (Pig. 5). This may be considered indirect evidence for a tight coupling between the
negatively
stained
with
phosphotungstic
two activities. In fact, it has been suggested that ADP-ATP exchange is due t)o t,he presence of a phosphorylated intermediat,e. formed during the process of calcium uptake (6).
FIG. 8. Fragmented sarcoplasmic membrane protein) for 90 minutes inhibitor. Negative staining with
Both residual ATPase and ADP-AT1 exchange of digested membranes retain their requirement for calcium. One should then t,hink t,hat calcium must bind to the membrane t’o cause enzyme activation. However,
reticulum after digestion with trypsin (1 mg ‘%? mg at 25”. Digestion interrupted by the addition of tripsin phosphotungstic acid. X232,000.
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no ATP-dependent calcium uptahe can be determined in membranes digested for more than 10 minutes. It is possible that this fraction of calcium is too small to be measured, due to instability of the complex. It may well be t’hat in nondigested vesicles a greater amount of calcium is bound to the carrier, in equilibrium with the high intravesicular concentration of the cation. Digested membrane fragments can bind a certain amount of calcium, independently of the presence of ATP (Figs. 4 and 6). We have no way of telling whether this calcium binding is a nonspecific phenomenon, or is a requirement for enzyme activation. A careful study on the cation binding sites available in fragmented sarcoplasmic reticulum has been recently published by Carvalho and Leo (16). Chloroform-methanol extracts of sarcoplasmic membranes were also found to have calcium binding capacity which could be competitively inhibited by other cation (17). A remarkable structural feature of control sarcoplasmic vesicles, as outlined by negative staining, is the presence of an outer granular layer. Elementary particles on the surface of mitochondrial membranes were originally delscribed by Fernandez-Moran (18). The mitochondrial particles are much larger than the granules observed on sarcoplasmic membranes, and can be easily recognized. On the other hand, Cunningham and Crane reported that different types of membranes, other than mitochondrial, are not really smooth, but present granular material on the outer surface (19). Observations on the granular appearance of microsomal membranes obtained from beef liver were reported by Maclennan et al. (20). Green et ar!. have defined “detachable sectors” that can be removed from the “base pieces” without loss of the basic membrane structure (21). The external granular layer of sarcoplasmic reticulum is completely removed by a short trypsin digestion (lo-20 minutes). This timing also produces inhibition of calcium accumulation and activation of ATPase activity. The available information does not allow speculation on whether these structural alterations and functional modifications are causally related or are simply
477
RETICULUM
produced at the important fact activity remains layer, which is digestion.”
same time by trypsin. One is that a high ATPase with the basal membrane more resistant to trypsin
ACKNOWLEDGMENTS The authors express their gratitude to Professors M. Morales and S. Wat,anabe for encouragement and frequent discussions, and to Professor W. Stoeckenius for very helpful comments. Miss Carolyn Piper (Univ. Calif. Berkeley) participated in some experiments as a summer student. REFERENCES 1. EBASHI, S. AXD LIPMAN, F., J. Cell Biol. 14, 389 (1962). 2. HASSELBACH, W. AND MAKINOSE, Biochem. Z. 333, 518 (1962); 339, 94 (1963). 3. WEBER, A., HERZ, R. AND REISS, I., J. Gen. Physiol. 46, 679 (1963). 4. MARTONOSI, A. AND FERETOS, R., J. Biol. Chem. 239, 648 (1964) ; 239, 659 (1964). 5. MUSCATELLO, U. et al., J. Biophys. Biochem. Cytol. 10, 201 (1961). 6. HASSELBACK, W., Progress Biophysics and Mol. Biol. 14, 167 (1964). 7. MARTONOSI, A., Fed. Proc. 23, 913 (1964). 8. NAGAI, T., MAKINOSE, M., AND HASSELBACH, W., Biochim. Biophys. Acta 43,223 (1960). 9. INESI, G. AND WATANABE, S., Arch. Biochem. Biophys. 121, 665 (1967). 10. INESI, G., GOODMAN, J., AND WATANABE, S., J. Biol. Chem. 242,4637 (1967). 11. MISSON, G., Chemiker Z. 32, 633 (1908). 12. LECOCQ, J. AND INESI, G., Analyt. Biochem. 16, 160 (1966). 13. MARCHESI, V. T. AND PALADE, G. E., Proc. Mat. Acad. Sci. 68, 991 (1967). 14. ODA, T. AND SEKI, S., 6th Internat. Congress for Electron Microscopy. Marusen Co., Ltd. Nihonbashi, Tokyo, page 387, (1966). 15. HASSELBACH, W. AND ELFVIN, L. G., J. Ultrastrut. Res. 17, 598 (1967). 16. CARVALHO, A. P. AND LEO, B., J. Gen. Physiol. 60, 1327 (1967). 17. INESI, G. AND WATANABE, S., Fed. Proc. 24, 208 (1965). 18. FERNANDEZ-MORAN, H., Circulation 26, 1039 (1962). 19. CUMINGHAM, W. P. AND CRANE, F. L. Exptl. Cell Biol. 44, 31 (1966). 20. MACLENNAN, D. H. et al., B.B.A. 131,59 (1967). 21. GREEN, D. E. et al., Arch. Biochem. Biophys. 119, 312 (1967). 6 After submitting this manuscript, an article by A. Martonosi appeared (J.BioZ. Chem. 243, 71, 1968), where experiments on trypsin digestion of sarcoplasmic reticulum are also described.