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Orientation of the β subunit polypeptide of (Na+ + K+)ATPase in the cell membrane
136
Biochimica et Biophvsica Acta 873 (1986) 136-142 Elsevier
BBA 32608
O r i e n t a t i o n o f t h e ,8 s u b u n i t p o l y p e p t i d e o f ( N a + + K + ) A T P a s e in t h e cell m e m b r a n e R o b e r t A. F a r l e y , R i c h a r d P. M i l l e r a n d A y a K u d r o w Department of Physiology and Biophysics, University of Southern California School of Medicine, 2025 Zonal A re., Los Angeles, CA 90033 (U.S.A.) (Received January 6th, 1986) (Revised manuscript received May 21st, 1986)
Key words: (Na + + K + )-ATPase; /~-subunit orientation; Subunit interaction; Glycosylation site; Immunochemistry; Proteolytic cleavage
Although the animal cell ( N a + + K +)-ATPase is composed of two polypeptide subunits, a and fl, v e ~ little is known about the fl subunit. In order to obtain information about the structure of this polypeptide, the subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the ~ subunit of (Na + + K +)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the ,8 subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-1fibeled ,8 subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, hut after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na + + K +)-ATPase for anti-fl subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the/~ subunit. These data indicate that the ~ subunit of ( N a + + K +)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.
Introduction The ( N a + + K+)-ATPase is composed of two polypeptide subunits, an a subunit with a mass of about 100 kDa, and a fl subunit with a mass of about 50 kDa [1,2]. The /3 subunit is a sialoglycoprotein. Although the structure of the a subunit Abbreviation: PMSF, phenylmethylsulfonyl fluoride.
has been the subject of numerous investigations, very little is known about the structure of the /3 subunit. Immunological analysis suggests that /3 subunits from different tissues or species may be structurally different [3], despite similarities in structure of the a subunits in the same preparations. The role of the fl subunit in (Na + + K+) ATPase activity is also not known. It has been suggested, however, that the /3 subunit may be
0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
137
important during the assembly of ( N a + + K+) ATPase in the cell membrane [3]. Since both the a subunit and the/3 subunit are synthesized coordinately [4], and are induced at the same time by thyroid hormone [5], interactions between the a subunit and the/3 subunit may be important for ( N a + + K + ) - A T P a s e assembly or function. In order to initiate an investigation of the interactions between the a subunit and the/3 subunit of (Na + + K+)-ATPase, the structure of the/3 subunit has been examined in these experiments. The ( N a + + K+)-ATPase was cleaved by chymotrypsin to generate large fragments of the/3 subunit, and the orientation of these fragments in the cell membrane was determined by examination of the cleavage in right-side-out vesicles containing the ( N a + + K+)-ATPase in a single orientation. The results of the investigations indicate that the /3 subunit inserts into the cell membrane near only one end of the polypeptide, and has at least two oligosaccharide chains exposed on the extracellular surface of the membrane.
Experimental procedures Purification of (Na + + K +)-ATPase. (Na + + K+)-ATPase was purified from dog kidney outer medulla by the method of Jorgensen [6]. Right-side out vesicles were prepared as described by Forbush [7]. Detergent activation of ( N a + + K+)ATPase in these vesicles was typically greater than 20-fold. The fl subunit of ( N a + + K+)-ATPase was purified by dissolving the purified ( N a + + K+)-ATPase in a solution of 2% (w/v) SDS, 50 mM Tris-HC1 (pH 8.0), 1 mM Na2EDTA, 1% ( v / v ) 2-mercaptoethanol, and separating the a subunit and the /3 subunit on a column of Sepharose CL-4B (5 × 120 cm). The pools of fl subunit fractions were lyophilized and dialyzed exhaustively against 0.2% (w/v) SDS, 25 mM imidazole-HC1 (pH 7.4), 1 mM Na2EDTA. SDS was removed from the fl subunit polypeptide on a column of AG1-X8 [8]. Proteolytic cleavage of (Na + + K +)-A TPase. 100 /~g of purified (Na + + K+)-ATPase or 100 /~g of membrane protein in right-side-out vesicles were incubated with 5 mM MgC12, 1 mM ouabain, 25 mM imidazole-HC1 (pH 7.4) for 5 min at 37°C. Chymotrypsin (0.5-10 ~g) was added and the
reaction was continued at 37 °C. At different times an aliquot was withdrawn from the reaction and the chymotrypsin reaction was stopped by the addition of 1 mM PMSF. The samples were frozen until all samples were collected, and then thawed by the addition of 1 mM PMSF. The reactions were centrifuged at 220 000 × g for 90 rain and the supernatants were removed and lyophilized. The proteins and peptides in the lyophilized supernatants and the pellets were separated on 12.5% polyacrylamide gels [9]. Trypsin digests were performed similarly except that the reactions were stopped with soybean trypsin inhibitor. Papain digests were performed according to Chin [15]. Carbohydrate labeling. The sialic acid residues on the oligosaccharide chains of the t3 subunit were oxidized with sodium periodate and reduced with NaB3H4 as described by Steck and Dawson [10]. Membrane-bound (Na + + K+)-ATPase (1 m g / m l ) in 50 mM sodium acetate, pH 5, was oxidized by 1 mM NalO 4 in the dark on ice for 15 rain. An equal volume of 100 mM NaAsO 2 in 50 mM sodium phosphate, pH 9.2, was added and the membranes were washed twice by centrifugation. The (Na + + K+)-ATPase was reduced by NaB3H4 (1.74 mCi; 0.2 mM) at room temperature for 10 min in 50 mM sodium phosphate, pH 9.2, and the reaction was stopped by the addition of an equal volume of 100 mM sodium acetate, pH 5. The sample was washed by centrifugation and suspended in 25 mM imidazole/HC1 (pH 7.4), 1 mM Na2EDTA. Neuraminidase and galactose oxidase reactions were performed exactly as described by Chin [15]. Tritium fluorography was performed using EN3HANCE (New England Nuclear). Immunoblots. Antisera against (Na + + K+) ATPase were prepared in rabbits as described by Farley et al. [11]. Antibodies against the fl subunit were purified from these antisera by chromatography of the antisera on an a-subunit affinity column [11], and the flow-through from this column was then incubated with diazophenylthioether paper [12] to which had been attached purified fl subunit. The anti-/3-subunit antibodies were eluted from this paper with 0.2 M glycine-HC1, pH 2.3, and immediately neutralized with 2 M Tris-HC1, pH 9.0. Electrophoretic transfer of proteins from polyacrylamide gels to the paper, and incubation
138 with anti-/3-subunit antibodies, was d o n e as described before [11].
A
B
C
Results and Discussion ap Detection of p r o t e o l y t i c fragments of the /3 subunit of ( N a + + K + ) - A T P a s e is c o m p l i c a t e d by the fact that there are no ligands or inhibitors of ( N a ~ + K + ) - A T P a s e activity that specifically b i n d to the /~ subunit. Protein staining after electrop h o r e t i c s e p a r a t i o n of cleavage p r o d u c t s does not d i s c r i m i n a t e between fragments of the a subunit and fragments of the fl subunit, a n d p r o t e o l y t i c cleavage of the ( N a + + K + ) - A T P a s e is k n o w n to generate several m a j o r fragments from the a subunit of the enzyme [13]. The fl subunit of ( N a + + K + ) - A T P a s e is a sialoglycoprotein, however, a n d o x i d a t i o n of the terminal sialic acid residues followed by reduction with tritiated b o r o h y d r i d e introduces a tritium label into the c a r b o h y d r a t e . The fate of these groups after proteolytic cleavage can then be followed without a m b i g u i t y due to the absence of c a r b o h y d r a t e on fragments from the a subunit. Fig. 1 shows the results of c h y m o t r y p s i n cleavage of the ( N a + + K + ) - A T P a s e after labeling the sialic acid residues of t h e / ~ subunit with tritiated b o r o h y d r i d e . Of the two subunits of ( N a + + K + ) ATPase, only the 58 k D a /3 subunit is labeled by this procedure. T h e r e is no label in the 94 k D a a subunit, b u t a labeled b a n d with a mass of a b o u t 150 k D a is also seen in the s a m p l e (lane A). Since this b a n d also reacts with b o t h a n t i - a - s u b u n i t a n t i b o d i e s a n d anti-fl-subunit a n t i b o d i e s ( d a t a not shown), it is p r o b a b l y an a/3 complex, a n d it a p p e a r s that the f o r m a t i o n of this complex is the result of boiling the s a m p l e before electrophoresis. A f t e r cleavage of the b o r o h y d r i d e - l a b e l e d ( N a + + K + ) - A T P a s e with c h y m o t r y p s i n in the presence of MgCI 2 a n d o u a b a i n , a 40 k D a fragment of t h e / ~ subunit is generated (lane B). This cleavage reaction is not d e p e n d e n t u p o n ouabain, a n d will occur in 5 m M MgC12 alone. In the absence of MgC12, however, c h y m o t r y p s i n cleaves t h e / 3 subunit very slowly (lane C). A second cleavage p r o d u c t expected from the reaction between c h y m o t r y p s i n and the/3 subunit of ( N a + + K + ) - A T P a s e is a fragment with an a p p a r e n t m o l e c u l a r weight of a b o u t 18000. In
o
p
Fig. 1. Labeling of carbohydrate residues on the /3 subunit of (Na + + K + )-ATPase. Purified (Na + + K + )-ATPase (1 mg) was suspended in 1 ml of 100 mM sodium acetate, pH 5, and 1 ml of 2 mM sodium periodate was added. The sample was incubated on ice in the dark for 15 rain before addition of 2 ml of 50 mM sodium arsenite in 50 mM sodium phosphate, pH 9.2. The membranes containing (Na + + K + )-ATPase were separated from the reagents by centrifugation and were suspended in 0.9 ml of 50 mM sodium phosphate, pH 9.2. 0.1 ml of 10 mM tritiated sodium borohydride was added and the sample was incubated for 10 rain at room temperature before the addition of 2 ml of 100 mM sodium acetate, pH 5, to stop the reaction. The membranes were washed twice by centrifugation and suspended in 0.15 ml of 25 mM imidazole-HCl, 1 mM Na 2EDTA (lane A). Chymotrypsin cleavage was performed in 5 mM magnesium chloride, 1 mM ouabain (lane B), or in 25 mM imidazole-HCl, 1 mM Na2EDTA (lane C) at a chymotrypsin/ATPase weight ratio of 1 : 1. After the reaction was stopped with 1 mM PMSF, the samples were separated on 10% SDS-polyacrylamide gels [9] and prepared for tritium fluorography.
several e x p e r i m e n t s in which p e r i o d a t e - o x i d i z e d b o r o h y d r i d e - l a b e l e d ( N a + + K + ) - A T P a s e was cleaved by c h y m o t r y p s i n this fragment was not o b s e r v e d on S D S - p o l y a c r y l a m i d e gels, but substantial a m o u n t s of r a d i o a c t i v i t y were found at the dye front. A l t h o u g h this suggests a more extensive proteolysis of this fragment, the p r o d u c tion of smaller p e p t i d e s is not a consequence of the c h y m o t r y p s i n reaction since the 18 k D a fragm e n t was detected using anti-/3-subunit a n t i b o d ies. This is illustrated in Fig. 2. In this e x p e r i m e n t ( N a + + K + ) - A T P a s e was not labeled with b o r o h y d r i d e before the c h y m o t r y p s i n reaction, but was i n c u b a t e d with anti-/~-subunit a n t i b o d i e s after transfer of the cleavage fragments to d i a z o p h e n y l thioether paper. The a n t i b o d i e s b i n d to only the fl subunit, a n d as the c h y m o t r y p s i n reaction proceeds, an i m m u n o r e a c t i v e fragment of a b o u t 22
139
0
01
05
Time 1
3
6
(hr)
94 K 68 K
Q 45K O7
21K 14K
Fig. 2. Cleavage of (Na + + K + )-ATPase by chymotrypsin and reaction with anti-/~-subunit antibodies. ( N a + + K + )-ATPase was cleaved by chymotrypsin for different times in 5 mM magnesium chloride, I mM ouabain as described in Fig. 1. Aliquots were removed from the reaction at the times indicated at the top of the figure and the membranes were separated from the soluble material by centrifugation. The supernatants were lyophilized and dissolved in 50 ffl of electrophoresis buffer [9]. The pellets were also dissolved in 50 ffl of the same buffer, and both samples were applied to 10% SDS-polyacrylamide gels. After electrophoresis, the gels were blotted to diazotized paper and incubated with anti-fl-subunit antibodies [11]. In the figure each time point is represented by two lanes on the gel containing either the pellet (left lane) or the supernatant (right lane).
kDa is generated. This fragment is gradually converted to a fragment of about 18 kDa. Integration of the blackened bands on the film indicates that the total amount of radioactivity found in the fl-subunit band and in these lower molecular weight bands is constant, after correction for the different efficiencies of transfer of the fragments and fl from the gel to the diazotized paper. This means that the 18 kDa fragment contains all of the sites on the/3 subunit that are recognized by antibodies. In this experiment the membranes containing ( N a + + K + ) - A T P a s e were separated from the reaction buffer by high-speed centrifugation after chymotrypsin cleavage. It can also be seen in this figure that the 18 kDa fragment of the fl subunit is retained in the membrane after it is generated, indicating that it interacts strongly with the hydrocarbon core of the lipid bilayer. The radioactivity in the supernatant lanes of this figure at long times of digestion is the result of incom-
plete sedimentation of the membranes during centrifugation. The combined molecular weights of the two fragments identified after chymotrypsin cleavage of the fl subunit are very nearly equal to the molecular weight of the intact fl subunit as estimated in this gel system. Although these molecular weights are only accurate to within about 10%, this observation suggests that the 40 kDa fragment and the 18 kDa fragment are only products of the cleavage reaction. Like the 18 kDa fragment, the 40 kDa fragment is retained in the membrane after chymotrypsin cleavage. The 40 kDa fragment does not span the lipid bilayer, however, since it can be removed from the membrane after cleavage by treatment with 0.1 M NaOH. This is illustrated in Fig. 3. In this experiment the membrane-bound, borohydride-labeled ( N a + + K+)-ATPase was first
Extraction: none
DTT
NaOH
Fig. 3. Extraction of membranes containing chymotrypsincleaved (Na + + K ÷ )-ATPase with dithiothreitol or sodium hydroxide. The carbohydrate on the fl subunit was labeled with tritiated borohydride as described in Fig. 1, and the (Na ÷ + K + )-ATPase was cleaved by chymotrypsin for 2 h at 37°C. The sample was divided into three parts that were extracted with either 0.1 M dithiothreitol (DTT), 0.1 M NaOH, 1 mM Na2EDTA (NaOH), or held on ice (None). The sample extracted with dithiothreitol was incubated at 37°C for 30 min before centrifugation at 220000 × g for 60 min, and the sample extracted with NaOH was centrifuged immediately after addition of 4 vol. of extraction solution at 4°C. The supernatant from each sample was lyophilized and was then dissolved in 50 ~tl of electrophoresis buffer [9] and the pellet was dissolved in the same buffer. After electrophoresis a fluorogram was prepared. A pair of lanes for each fraction is shown, with the extracted pellet on the left and the extracted supernatant on the right.
140
cleaved with chymotrypsin, and then, after the membranes were recovered by centrifugation, they were extracted with either 0.1 M dithiothreitol for 30 rain at 37°C, or 0.1 M NaOH, 1 mM Na2EDTA. The membranes were separated from supernatants by centrifugation and both membrane and supernatant fractions were analyzed by SDS-polyacrylamide gel electrophoresis. Before treatment with NaOH both the intact fl subunit and the 40 kDa fragment were recovered in the membrane pellet, whereas after extraction with NaOH, the 40 kDa fragment was found in the supernatant. Extraction with dithiothreitol alone did not release the 40 kDa fragment from the membrane. Fig. 4 shows that the site of cleavage of the/~ subunit by chymotrypsin is on the extracellular side of the membrane. In this experiment the ( N a + + K+)-ATPase was prepared in right-sideout vesicles [7] that are tight to substrate ATP. The ATPase activity of these vesicles was activated 20-30-fold by detergent. Exposure of these vesicles to chymotrypsin generated the same fragments of Time 0
5
15
30 (rain)
P 40K
18K
Fig. 4. Cleavage of tight right-side-out vesicles containing (Na + + K + ) - A T P a s e by chymotrypsin. Tight right-side-out vesicles were prepared according to Forbush [7]. 500 fig of vesicle protein was incubated with 50 fig of chymotrypsin in 5 mM magnesium chloride, 1 m M ouabain for the times indicated at the top of the figure. At the indicated times the samples were separated into pellet and supernatant fractions and prepared for electrophoresis as described in Fig. 3. The gel was blotted and incubated with anti-fl-subunit antibodies as described in Fig. 2. The pellet fraction is the left lane of each pair of lanes at each time,
the fl subunit that were generated by cleavage of the purified ATPase in membrane sheets. The figure demonstrates the appearance of the 18 kDa fragment that is recognized by anti-/~-subunit antibodies as the time of reaction with chymotrypsin is increased. Immunological analysis of the chymotrypsin-cleaved vesicles with anti-a-subunit antibodies indicated that the a subunit was not cleaved in this reaction. Since the cleavage sites for chymotrypsin on the a subunit are located on the cytoplasmic surface of the membrane [11], this indicates that the cleavage site on the fl subunit for chymotrypsin is located on the outside surface of the vesicles. In order to determine the spatial relationship between the two fragments generated from the fl subunit by chymotrypsin, the borohydride-labeled ]3 subunit was crosslinked with o-phenanthroline/ CuSO 4 and then cleaved by chymotrypsin. The crosslinked products were separated on two-dimensional SDS-polyacrylamide gels in which reducing agent was absent in the first dimension but present in the second dimension [16]. The results of this procedure indicated that the 40 kDa fragment was crosslinked in the first dimension to a smaller fragment of about 18 kDa, and suggested that sulfhydryl groups may be located within the ,8 subunit in such locations to permit intramolecular crosslink formation between the two products of the chymotrypsin reaction. This was confirmed by the experiment presented in Fig. 5, in which the proteolytic fragments of borohydride labeled, chymotrypsin-cleaved (Na + + K +)-ATPase were separated by SDS-polyacrylamide gel electrophoresis in either the absence or the presence of reducing agent. The absence of the reducing agent resulted in the co-migration of the cleaved fragments with the intact ]9 subunit, whereas the presence of the reducing agent separates the fragments so that they migrate independently in the gels. The ]3 subunit exposes most of its mass and all of its carbohydrate on the outside surface of the cell membrane [17]. The experiments reported here confirm this and also demonstrate that the fl subunit is anchored in the cell membrane in the region of the polypeptide from which the 18 kDa chymotryptic fragment is derived. The 40 kDa fragment is associated with the 18 kDa fragment
141 -2ME
+2ME
58K 40K
Fig. 5. Reduction of disulfide-linked chymotryptic fragments of the /3 subunit. ( N a + + K ÷ )-ATPase was labeled with tritiated sodium borohydride and was cleaved with chymotrypsin as described in Experimental procedures. After centrifugation at 220000)< g for 60 min the membranes were suspended in 25 m M imidazole-HCl, 1 m M N a 2 E D T A , pH 7.4. 34 /Lg protein were added to electrophoresis sample buffer [9] either without 2-mercaptoethanol ( - 2ME) or with 2-mercaptoethanol ( + 2ME). The samples were incubated at 3 7 ° C for 1 h before electrophoresis and fluorography.
through both electrostatic interactions and at least one disulfide bond but does not pepetrate the lipid bilayer. It is interesting to note that the immunoreactive part of the/3 subunit is associated with the membrane-embedded 18 kDa chymotryptic fragment, since it has previously been shown that the /3 subunits from different tissues are immunologically distinct [3]. If the /3 subunit is associated with the a subunit in a complex that is important for assembly of the (Na + + K +)-ATPase [3], it is likely that this interaction occurs on the cytoplasmic surface of the membrane .This is because most of the mass of the a subunit is on the cytoplasmic surface [11,14]. The antigenic differences seen with the /3 subunits from different ( N a + + K+)-ATPase sources may, therefore, reflect differences in this part of the polypeptide that have functional or regulatory consequences. During the preparation of this report the results of proteolytic fragmentation studies of the/3 subunit using papain were published by Chin [15]. In that report it was determined that a 40 kDa fragment was derived from the amino-terminal of the
fl subunit, and that a 16 kDa fragment was derived from the carboxy-terminal. The difference in molecular weights of the smaller fragments in these two reports is probably not significant. A model for the folding of the /3 subunit was also suggested by Chin in which the fl subunit folds through the cell membrane several times. That model is not consistent with either the data presented in this report or the predicted structure derived from the c D N A sequence of the/3 subunit from Torpedo [18]. Although the amino-terminal chymotryptic fragment has not been identified in this study, a comparison of these data with the predicted structure suggests that the 18 kDa fragment is derived from the amino-terminal of the fl subunit. The 18 kDa fragment is the only fragment that is embedded within the lipid bilayer, and the amino-terminal is the region predicted from the c D N A sequence to contain the only membrane-spanning sequences of the protein. Whether the polypeptide spans the membrane more than once in that region, however, has not been determined. In the study by Chin the fl subunit was labeled by NaB3H4 at galactose moieties after removal of the terminal sialic acid residues with neuraminidase, and it was observed that both the 40 kDa fragment and the 18 kDa fragment were labeled. This contrasts with the observation made here that only the 40 kDa fragment was labeled after oxidation with periodate. In order to resolve this discrepancy the fl subunit was labeled with tritiated borohydride in an additional experiment either after oxidation with periodate, or after removal of sialic acid residues with neuraminidase followed by treatment with galactose oxidase, and the cleavage with chymotrypsin was repeated. The results of this experiment confirm that both the 40 kDa fragment and the 18 kDa fragment contain oligosaccharide chains. This experiment also suggests that the failure to detect a borohydride-labeled 18 kDa fragment after periodate oxidation is probably due to degradation of the fragment during the chemical modification procedure. The production of the 40 kDa fragment and the 18 kDa fragment could be obtained after cleavage of the/3 subunit with either chymotrypsin, trypsin, papain or subtilisin. These observations suggest that there is a region of the fl subunit that is
142 extraordinarily sensitive to proteolytic cleavage, perhaps due to the folding of the polypeptide. This is supported by the observation that in detergent solution the /3 s u b u n i t is cleaved by these proteinases at multiple sites, resulting in the generation of m a n y small proteolytic fragments. Thus, the reported resistance of the/3 s u b u n i t to proteolytic cleavage is not due to the absence of sensitive amide bonds, but is the result of the folding of the polypeptide.
Acknowledgements This work was supported by grant n u m b e r PCM-8409127 from the N a t i o n a l Science F o u n d a tion, and was done d u r i n g the tenure of an Established Investigatorship from the A m e r i c a n Heart Association to R.A.F. with funds c o n t r i b u t e d in part by the A m e r i c a n Heart Association, Greater Los Angeles Affiliate. R.P.M. is an N I H predoctoral fellow at the University of Southern California supported by the N I H training grant T32GM08017.
References 1 Jorgensen, P.L. (1982) Biochim. Biophys. Acta 694, 27-68 2 Kyte, J. (1972) J. Biol. Chem. 247, 7642-7649 3 Hiatt, A., McDonough, A.A. and Edelman, I.S. (1984) J. Biol. Chem. 259, 2629-2635 4 Churchill, L. and Hokin, L.E. (1976) Biochim. Biophys. Acta 434, 258-264 5 Lo, C.S. and Lo, T.N. (1980) J. Biol. Chem. 255, 2131-2136 6 Jorgensen, P.L. (1974) Biochim. Biophys. Acta 356, 53-67 7 Forbush, B., III (1982) J. Biol. Chem. 257, 12678-12684 8 Lenard, J. (1971) Biochem. Biophys. Res. Commun. 45, 662-667 9 Laemmli, U.K. (1970) Nature 227, 680-685 10 Steck, T.L. and Dawson, G. (1974) J. Biol. Chem. 249, 2135 2142 11 Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906 12 Seed, B. (1982) Nucleic Aids Res. 10, 1799-1810 13 Jorgensen, P.L. and Farley, R.A. (1986) Methods Enzymol., in the press 14 Nicholas, R.A. (1984) Biochemistry23, 888-898 15 Chin, G.J. (1985) Biochemistry 24, 5943-5947 16 Wang, K. and Richards, F.M. (1974) J. Biol. Chem. 249. 8005-8018 17 Dzhandzhugazyan, K. and Jorgensen, P.L. (1985) Biochim. Biophys. Acta 817, 165-173 18 Noguchi, S., Noda, M., Takahashi, H., Kawakami, K., Ohta, T., Nagano, K., Hirose, T., lnayama, S., Kawamura, M. and Numa, S. (1986) FEBS Lett. 196, 315-320
Biochimica et Biophvsica Acta 873 (1986) 136-142 Elsevier
BBA 32608
O r i e n t a t i o n o f t h e ,8 s u b u n i t p o l y p e p t i d e o f ( N a + + K + ) A T P a s e in t h e cell m e m b r a n e R o b e r t A. F a r l e y , R i c h a r d P. M i l l e r a n d A y a K u d r o w Department of Physiology and Biophysics, University of Southern California School of Medicine, 2025 Zonal A re., Los Angeles, CA 90033 (U.S.A.) (Received January 6th, 1986) (Revised manuscript received May 21st, 1986)
Key words: (Na + + K + )-ATPase; /~-subunit orientation; Subunit interaction; Glycosylation site; Immunochemistry; Proteolytic cleavage
Although the animal cell ( N a + + K +)-ATPase is composed of two polypeptide subunits, a and fl, v e ~ little is known about the fl subunit. In order to obtain information about the structure of this polypeptide, the subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the ~ subunit of (Na + + K +)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the ,8 subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-1fibeled ,8 subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, hut after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na + + K +)-ATPase for anti-fl subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the/~ subunit. These data indicate that the ~ subunit of ( N a + + K +)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.
Introduction The ( N a + + K+)-ATPase is composed of two polypeptide subunits, an a subunit with a mass of about 100 kDa, and a fl subunit with a mass of about 50 kDa [1,2]. The /3 subunit is a sialoglycoprotein. Although the structure of the a subunit Abbreviation: PMSF, phenylmethylsulfonyl fluoride.
has been the subject of numerous investigations, very little is known about the structure of the /3 subunit. Immunological analysis suggests that /3 subunits from different tissues or species may be structurally different [3], despite similarities in structure of the a subunits in the same preparations. The role of the fl subunit in (Na + + K+) ATPase activity is also not known. It has been suggested, however, that the /3 subunit may be
0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
137
important during the assembly of ( N a + + K+) ATPase in the cell membrane [3]. Since both the a subunit and the/3 subunit are synthesized coordinately [4], and are induced at the same time by thyroid hormone [5], interactions between the a subunit and the/3 subunit may be important for ( N a + + K + ) - A T P a s e assembly or function. In order to initiate an investigation of the interactions between the a subunit and the/3 subunit of (Na + + K+)-ATPase, the structure of the/3 subunit has been examined in these experiments. The ( N a + + K+)-ATPase was cleaved by chymotrypsin to generate large fragments of the/3 subunit, and the orientation of these fragments in the cell membrane was determined by examination of the cleavage in right-side-out vesicles containing the ( N a + + K+)-ATPase in a single orientation. The results of the investigations indicate that the /3 subunit inserts into the cell membrane near only one end of the polypeptide, and has at least two oligosaccharide chains exposed on the extracellular surface of the membrane.
Experimental procedures Purification of (Na + + K +)-ATPase. (Na + + K+)-ATPase was purified from dog kidney outer medulla by the method of Jorgensen [6]. Right-side out vesicles were prepared as described by Forbush [7]. Detergent activation of ( N a + + K+)ATPase in these vesicles was typically greater than 20-fold. The fl subunit of ( N a + + K+)-ATPase was purified by dissolving the purified ( N a + + K+)-ATPase in a solution of 2% (w/v) SDS, 50 mM Tris-HC1 (pH 8.0), 1 mM Na2EDTA, 1% ( v / v ) 2-mercaptoethanol, and separating the a subunit and the /3 subunit on a column of Sepharose CL-4B (5 × 120 cm). The pools of fl subunit fractions were lyophilized and dialyzed exhaustively against 0.2% (w/v) SDS, 25 mM imidazole-HC1 (pH 7.4), 1 mM Na2EDTA. SDS was removed from the fl subunit polypeptide on a column of AG1-X8 [8]. Proteolytic cleavage of (Na + + K +)-A TPase. 100 /~g of purified (Na + + K+)-ATPase or 100 /~g of membrane protein in right-side-out vesicles were incubated with 5 mM MgC12, 1 mM ouabain, 25 mM imidazole-HC1 (pH 7.4) for 5 min at 37°C. Chymotrypsin (0.5-10 ~g) was added and the
reaction was continued at 37 °C. At different times an aliquot was withdrawn from the reaction and the chymotrypsin reaction was stopped by the addition of 1 mM PMSF. The samples were frozen until all samples were collected, and then thawed by the addition of 1 mM PMSF. The reactions were centrifuged at 220 000 × g for 90 rain and the supernatants were removed and lyophilized. The proteins and peptides in the lyophilized supernatants and the pellets were separated on 12.5% polyacrylamide gels [9]. Trypsin digests were performed similarly except that the reactions were stopped with soybean trypsin inhibitor. Papain digests were performed according to Chin [15]. Carbohydrate labeling. The sialic acid residues on the oligosaccharide chains of the t3 subunit were oxidized with sodium periodate and reduced with NaB3H4 as described by Steck and Dawson [10]. Membrane-bound (Na + + K+)-ATPase (1 m g / m l ) in 50 mM sodium acetate, pH 5, was oxidized by 1 mM NalO 4 in the dark on ice for 15 rain. An equal volume of 100 mM NaAsO 2 in 50 mM sodium phosphate, pH 9.2, was added and the membranes were washed twice by centrifugation. The (Na + + K+)-ATPase was reduced by NaB3H4 (1.74 mCi; 0.2 mM) at room temperature for 10 min in 50 mM sodium phosphate, pH 9.2, and the reaction was stopped by the addition of an equal volume of 100 mM sodium acetate, pH 5. The sample was washed by centrifugation and suspended in 25 mM imidazole/HC1 (pH 7.4), 1 mM Na2EDTA. Neuraminidase and galactose oxidase reactions were performed exactly as described by Chin [15]. Tritium fluorography was performed using EN3HANCE (New England Nuclear). Immunoblots. Antisera against (Na + + K+) ATPase were prepared in rabbits as described by Farley et al. [11]. Antibodies against the fl subunit were purified from these antisera by chromatography of the antisera on an a-subunit affinity column [11], and the flow-through from this column was then incubated with diazophenylthioether paper [12] to which had been attached purified fl subunit. The anti-/3-subunit antibodies were eluted from this paper with 0.2 M glycine-HC1, pH 2.3, and immediately neutralized with 2 M Tris-HC1, pH 9.0. Electrophoretic transfer of proteins from polyacrylamide gels to the paper, and incubation
138 with anti-/3-subunit antibodies, was d o n e as described before [11].
A
B
C
Results and Discussion ap Detection of p r o t e o l y t i c fragments of the /3 subunit of ( N a + + K + ) - A T P a s e is c o m p l i c a t e d by the fact that there are no ligands or inhibitors of ( N a ~ + K + ) - A T P a s e activity that specifically b i n d to the /~ subunit. Protein staining after electrop h o r e t i c s e p a r a t i o n of cleavage p r o d u c t s does not d i s c r i m i n a t e between fragments of the a subunit and fragments of the fl subunit, a n d p r o t e o l y t i c cleavage of the ( N a + + K + ) - A T P a s e is k n o w n to generate several m a j o r fragments from the a subunit of the enzyme [13]. The fl subunit of ( N a + + K + ) - A T P a s e is a sialoglycoprotein, however, a n d o x i d a t i o n of the terminal sialic acid residues followed by reduction with tritiated b o r o h y d r i d e introduces a tritium label into the c a r b o h y d r a t e . The fate of these groups after proteolytic cleavage can then be followed without a m b i g u i t y due to the absence of c a r b o h y d r a t e on fragments from the a subunit. Fig. 1 shows the results of c h y m o t r y p s i n cleavage of the ( N a + + K + ) - A T P a s e after labeling the sialic acid residues of t h e / ~ subunit with tritiated b o r o h y d r i d e . Of the two subunits of ( N a + + K + ) ATPase, only the 58 k D a /3 subunit is labeled by this procedure. T h e r e is no label in the 94 k D a a subunit, b u t a labeled b a n d with a mass of a b o u t 150 k D a is also seen in the s a m p l e (lane A). Since this b a n d also reacts with b o t h a n t i - a - s u b u n i t a n t i b o d i e s a n d anti-fl-subunit a n t i b o d i e s ( d a t a not shown), it is p r o b a b l y an a/3 complex, a n d it a p p e a r s that the f o r m a t i o n of this complex is the result of boiling the s a m p l e before electrophoresis. A f t e r cleavage of the b o r o h y d r i d e - l a b e l e d ( N a + + K + ) - A T P a s e with c h y m o t r y p s i n in the presence of MgCI 2 a n d o u a b a i n , a 40 k D a fragment of t h e / ~ subunit is generated (lane B). This cleavage reaction is not d e p e n d e n t u p o n ouabain, a n d will occur in 5 m M MgC12 alone. In the absence of MgC12, however, c h y m o t r y p s i n cleaves t h e / 3 subunit very slowly (lane C). A second cleavage p r o d u c t expected from the reaction between c h y m o t r y p s i n and the/3 subunit of ( N a + + K + ) - A T P a s e is a fragment with an a p p a r e n t m o l e c u l a r weight of a b o u t 18000. In
o
p
Fig. 1. Labeling of carbohydrate residues on the /3 subunit of (Na + + K + )-ATPase. Purified (Na + + K + )-ATPase (1 mg) was suspended in 1 ml of 100 mM sodium acetate, pH 5, and 1 ml of 2 mM sodium periodate was added. The sample was incubated on ice in the dark for 15 rain before addition of 2 ml of 50 mM sodium arsenite in 50 mM sodium phosphate, pH 9.2. The membranes containing (Na + + K + )-ATPase were separated from the reagents by centrifugation and were suspended in 0.9 ml of 50 mM sodium phosphate, pH 9.2. 0.1 ml of 10 mM tritiated sodium borohydride was added and the sample was incubated for 10 rain at room temperature before the addition of 2 ml of 100 mM sodium acetate, pH 5, to stop the reaction. The membranes were washed twice by centrifugation and suspended in 0.15 ml of 25 mM imidazole-HCl, 1 mM Na 2EDTA (lane A). Chymotrypsin cleavage was performed in 5 mM magnesium chloride, 1 mM ouabain (lane B), or in 25 mM imidazole-HCl, 1 mM Na2EDTA (lane C) at a chymotrypsin/ATPase weight ratio of 1 : 1. After the reaction was stopped with 1 mM PMSF, the samples were separated on 10% SDS-polyacrylamide gels [9] and prepared for tritium fluorography.
several e x p e r i m e n t s in which p e r i o d a t e - o x i d i z e d b o r o h y d r i d e - l a b e l e d ( N a + + K + ) - A T P a s e was cleaved by c h y m o t r y p s i n this fragment was not o b s e r v e d on S D S - p o l y a c r y l a m i d e gels, but substantial a m o u n t s of r a d i o a c t i v i t y were found at the dye front. A l t h o u g h this suggests a more extensive proteolysis of this fragment, the p r o d u c tion of smaller p e p t i d e s is not a consequence of the c h y m o t r y p s i n reaction since the 18 k D a fragm e n t was detected using anti-/3-subunit a n t i b o d ies. This is illustrated in Fig. 2. In this e x p e r i m e n t ( N a + + K + ) - A T P a s e was not labeled with b o r o h y d r i d e before the c h y m o t r y p s i n reaction, but was i n c u b a t e d with anti-/~-subunit a n t i b o d i e s after transfer of the cleavage fragments to d i a z o p h e n y l thioether paper. The a n t i b o d i e s b i n d to only the fl subunit, a n d as the c h y m o t r y p s i n reaction proceeds, an i m m u n o r e a c t i v e fragment of a b o u t 22
139
0
01
05
Time 1
3
6
(hr)
94 K 68 K
Q 45K O7
21K 14K
Fig. 2. Cleavage of (Na + + K + )-ATPase by chymotrypsin and reaction with anti-/~-subunit antibodies. ( N a + + K + )-ATPase was cleaved by chymotrypsin for different times in 5 mM magnesium chloride, I mM ouabain as described in Fig. 1. Aliquots were removed from the reaction at the times indicated at the top of the figure and the membranes were separated from the soluble material by centrifugation. The supernatants were lyophilized and dissolved in 50 ffl of electrophoresis buffer [9]. The pellets were also dissolved in 50 ffl of the same buffer, and both samples were applied to 10% SDS-polyacrylamide gels. After electrophoresis, the gels were blotted to diazotized paper and incubated with anti-fl-subunit antibodies [11]. In the figure each time point is represented by two lanes on the gel containing either the pellet (left lane) or the supernatant (right lane).
kDa is generated. This fragment is gradually converted to a fragment of about 18 kDa. Integration of the blackened bands on the film indicates that the total amount of radioactivity found in the fl-subunit band and in these lower molecular weight bands is constant, after correction for the different efficiencies of transfer of the fragments and fl from the gel to the diazotized paper. This means that the 18 kDa fragment contains all of the sites on the/3 subunit that are recognized by antibodies. In this experiment the membranes containing ( N a + + K + ) - A T P a s e were separated from the reaction buffer by high-speed centrifugation after chymotrypsin cleavage. It can also be seen in this figure that the 18 kDa fragment of the fl subunit is retained in the membrane after it is generated, indicating that it interacts strongly with the hydrocarbon core of the lipid bilayer. The radioactivity in the supernatant lanes of this figure at long times of digestion is the result of incom-
plete sedimentation of the membranes during centrifugation. The combined molecular weights of the two fragments identified after chymotrypsin cleavage of the fl subunit are very nearly equal to the molecular weight of the intact fl subunit as estimated in this gel system. Although these molecular weights are only accurate to within about 10%, this observation suggests that the 40 kDa fragment and the 18 kDa fragment are only products of the cleavage reaction. Like the 18 kDa fragment, the 40 kDa fragment is retained in the membrane after chymotrypsin cleavage. The 40 kDa fragment does not span the lipid bilayer, however, since it can be removed from the membrane after cleavage by treatment with 0.1 M NaOH. This is illustrated in Fig. 3. In this experiment the membrane-bound, borohydride-labeled ( N a + + K+)-ATPase was first
Extraction: none
DTT
NaOH
Fig. 3. Extraction of membranes containing chymotrypsincleaved (Na + + K ÷ )-ATPase with dithiothreitol or sodium hydroxide. The carbohydrate on the fl subunit was labeled with tritiated borohydride as described in Fig. 1, and the (Na ÷ + K + )-ATPase was cleaved by chymotrypsin for 2 h at 37°C. The sample was divided into three parts that were extracted with either 0.1 M dithiothreitol (DTT), 0.1 M NaOH, 1 mM Na2EDTA (NaOH), or held on ice (None). The sample extracted with dithiothreitol was incubated at 37°C for 30 min before centrifugation at 220000 × g for 60 min, and the sample extracted with NaOH was centrifuged immediately after addition of 4 vol. of extraction solution at 4°C. The supernatant from each sample was lyophilized and was then dissolved in 50 ~tl of electrophoresis buffer [9] and the pellet was dissolved in the same buffer. After electrophoresis a fluorogram was prepared. A pair of lanes for each fraction is shown, with the extracted pellet on the left and the extracted supernatant on the right.
140
cleaved with chymotrypsin, and then, after the membranes were recovered by centrifugation, they were extracted with either 0.1 M dithiothreitol for 30 rain at 37°C, or 0.1 M NaOH, 1 mM Na2EDTA. The membranes were separated from supernatants by centrifugation and both membrane and supernatant fractions were analyzed by SDS-polyacrylamide gel electrophoresis. Before treatment with NaOH both the intact fl subunit and the 40 kDa fragment were recovered in the membrane pellet, whereas after extraction with NaOH, the 40 kDa fragment was found in the supernatant. Extraction with dithiothreitol alone did not release the 40 kDa fragment from the membrane. Fig. 4 shows that the site of cleavage of the/~ subunit by chymotrypsin is on the extracellular side of the membrane. In this experiment the ( N a + + K+)-ATPase was prepared in right-sideout vesicles [7] that are tight to substrate ATP. The ATPase activity of these vesicles was activated 20-30-fold by detergent. Exposure of these vesicles to chymotrypsin generated the same fragments of Time 0
5
15
30 (rain)
P 40K
18K
Fig. 4. Cleavage of tight right-side-out vesicles containing (Na + + K + ) - A T P a s e by chymotrypsin. Tight right-side-out vesicles were prepared according to Forbush [7]. 500 fig of vesicle protein was incubated with 50 fig of chymotrypsin in 5 mM magnesium chloride, 1 m M ouabain for the times indicated at the top of the figure. At the indicated times the samples were separated into pellet and supernatant fractions and prepared for electrophoresis as described in Fig. 3. The gel was blotted and incubated with anti-fl-subunit antibodies as described in Fig. 2. The pellet fraction is the left lane of each pair of lanes at each time,
the fl subunit that were generated by cleavage of the purified ATPase in membrane sheets. The figure demonstrates the appearance of the 18 kDa fragment that is recognized by anti-/~-subunit antibodies as the time of reaction with chymotrypsin is increased. Immunological analysis of the chymotrypsin-cleaved vesicles with anti-a-subunit antibodies indicated that the a subunit was not cleaved in this reaction. Since the cleavage sites for chymotrypsin on the a subunit are located on the cytoplasmic surface of the membrane [11], this indicates that the cleavage site on the fl subunit for chymotrypsin is located on the outside surface of the vesicles. In order to determine the spatial relationship between the two fragments generated from the fl subunit by chymotrypsin, the borohydride-labeled ]3 subunit was crosslinked with o-phenanthroline/ CuSO 4 and then cleaved by chymotrypsin. The crosslinked products were separated on two-dimensional SDS-polyacrylamide gels in which reducing agent was absent in the first dimension but present in the second dimension [16]. The results of this procedure indicated that the 40 kDa fragment was crosslinked in the first dimension to a smaller fragment of about 18 kDa, and suggested that sulfhydryl groups may be located within the ,8 subunit in such locations to permit intramolecular crosslink formation between the two products of the chymotrypsin reaction. This was confirmed by the experiment presented in Fig. 5, in which the proteolytic fragments of borohydride labeled, chymotrypsin-cleaved (Na + + K +)-ATPase were separated by SDS-polyacrylamide gel electrophoresis in either the absence or the presence of reducing agent. The absence of the reducing agent resulted in the co-migration of the cleaved fragments with the intact ]9 subunit, whereas the presence of the reducing agent separates the fragments so that they migrate independently in the gels. The ]3 subunit exposes most of its mass and all of its carbohydrate on the outside surface of the cell membrane [17]. The experiments reported here confirm this and also demonstrate that the fl subunit is anchored in the cell membrane in the region of the polypeptide from which the 18 kDa chymotryptic fragment is derived. The 40 kDa fragment is associated with the 18 kDa fragment
141 -2ME
+2ME
58K 40K
Fig. 5. Reduction of disulfide-linked chymotryptic fragments of the /3 subunit. ( N a + + K ÷ )-ATPase was labeled with tritiated sodium borohydride and was cleaved with chymotrypsin as described in Experimental procedures. After centrifugation at 220000)< g for 60 min the membranes were suspended in 25 m M imidazole-HCl, 1 m M N a 2 E D T A , pH 7.4. 34 /Lg protein were added to electrophoresis sample buffer [9] either without 2-mercaptoethanol ( - 2ME) or with 2-mercaptoethanol ( + 2ME). The samples were incubated at 3 7 ° C for 1 h before electrophoresis and fluorography.
through both electrostatic interactions and at least one disulfide bond but does not pepetrate the lipid bilayer. It is interesting to note that the immunoreactive part of the/3 subunit is associated with the membrane-embedded 18 kDa chymotryptic fragment, since it has previously been shown that the /3 subunits from different tissues are immunologically distinct [3]. If the /3 subunit is associated with the a subunit in a complex that is important for assembly of the (Na + + K +)-ATPase [3], it is likely that this interaction occurs on the cytoplasmic surface of the membrane .This is because most of the mass of the a subunit is on the cytoplasmic surface [11,14]. The antigenic differences seen with the /3 subunits from different ( N a + + K+)-ATPase sources may, therefore, reflect differences in this part of the polypeptide that have functional or regulatory consequences. During the preparation of this report the results of proteolytic fragmentation studies of the/3 subunit using papain were published by Chin [15]. In that report it was determined that a 40 kDa fragment was derived from the amino-terminal of the
fl subunit, and that a 16 kDa fragment was derived from the carboxy-terminal. The difference in molecular weights of the smaller fragments in these two reports is probably not significant. A model for the folding of the /3 subunit was also suggested by Chin in which the fl subunit folds through the cell membrane several times. That model is not consistent with either the data presented in this report or the predicted structure derived from the c D N A sequence of the/3 subunit from Torpedo [18]. Although the amino-terminal chymotryptic fragment has not been identified in this study, a comparison of these data with the predicted structure suggests that the 18 kDa fragment is derived from the amino-terminal of the fl subunit. The 18 kDa fragment is the only fragment that is embedded within the lipid bilayer, and the amino-terminal is the region predicted from the c D N A sequence to contain the only membrane-spanning sequences of the protein. Whether the polypeptide spans the membrane more than once in that region, however, has not been determined. In the study by Chin the fl subunit was labeled by NaB3H4 at galactose moieties after removal of the terminal sialic acid residues with neuraminidase, and it was observed that both the 40 kDa fragment and the 18 kDa fragment were labeled. This contrasts with the observation made here that only the 40 kDa fragment was labeled after oxidation with periodate. In order to resolve this discrepancy the fl subunit was labeled with tritiated borohydride in an additional experiment either after oxidation with periodate, or after removal of sialic acid residues with neuraminidase followed by treatment with galactose oxidase, and the cleavage with chymotrypsin was repeated. The results of this experiment confirm that both the 40 kDa fragment and the 18 kDa fragment contain oligosaccharide chains. This experiment also suggests that the failure to detect a borohydride-labeled 18 kDa fragment after periodate oxidation is probably due to degradation of the fragment during the chemical modification procedure. The production of the 40 kDa fragment and the 18 kDa fragment could be obtained after cleavage of the/3 subunit with either chymotrypsin, trypsin, papain or subtilisin. These observations suggest that there is a region of the fl subunit that is
142 extraordinarily sensitive to proteolytic cleavage, perhaps due to the folding of the polypeptide. This is supported by the observation that in detergent solution the /3 s u b u n i t is cleaved by these proteinases at multiple sites, resulting in the generation of m a n y small proteolytic fragments. Thus, the reported resistance of the/3 s u b u n i t to proteolytic cleavage is not due to the absence of sensitive amide bonds, but is the result of the folding of the polypeptide.
Acknowledgements This work was supported by grant n u m b e r PCM-8409127 from the N a t i o n a l Science F o u n d a tion, and was done d u r i n g the tenure of an Established Investigatorship from the A m e r i c a n Heart Association to R.A.F. with funds c o n t r i b u t e d in part by the A m e r i c a n Heart Association, Greater Los Angeles Affiliate. R.P.M. is an N I H predoctoral fellow at the University of Southern California supported by the N I H training grant T32GM08017.
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