Biochemical properties of purified transverse tubules isolated from skeletal muscle triads

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 260, No. 1, January, pp. l-9,1988

Biochemical Properties of Purified Transverse Tubules Isolated from Skeletal Muscle Triads’ DOUGLAS J. HORGAN2 CSIRO Division

AND

RONALD KUYPERS

of Food Research, Meat Research Laboratory, P.O. Box 12, Cannon Hill, Queensland 4170, Australia

Received June 11,198’7, and in revised form August 31, 1987

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104,70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [T-~~P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out. 0 1988 Academic

Press. Inc

In order to resolve the mechanism of excitation-contraction coupling in skeletal muscle, it is important to separate and characterize all the various membrane components. Of these, the t-tubular3 system is responsible for the conduction of the nerve impulse from the surface of the muscle cell to the terminal cisternae, which are specialized elements of the SR involved in the release of internal Ca2+ (for review see Ref. (1)). To date two structural elements of the t-tubular system have been identified. The first of these are the “junctional elements,” i.e., those parts

of the t-tubular system which are opposed between two terminal cisternae of the SR. In addition, there are the “free elements” which are not physically associated with the terminal cisternae (1). Junctional ttubules have until recently been prepared solely by a French press treatment which causes a mechanical disruption of the triad, thus freeing the t-tubules (2). Free t-tubules have been prepared by sucrosegradient centrifugation of a light microsomal fraction after contaminating SR vesicles had been loaded with calcium phosphate (3). The junctional and free ttubules differ in their biochemical properties, e.g., their M$+-ATPase activities (4, 5). It has been postulated that these differences could be due to the severe treatment of the French press (3, 6). A milder procedure for the isolation of junctional t-tubules based on the dissociation of triads in ion-free sucrose gradients has been recently developed (7). In this paper the further purification and biochemical characterization of these t-tubules is described.

r This research was supported in part by the Australian Meat and Livestock Research and Development Corporation. ’ To whom correspondence should be addressed. 3 Abbreviations used: t-tubules, transverse tubules; SR, sarcoplasmic reticulum; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(P-aminoethyl ether)-N,N’-tetraacetic acid; TCA, trichloroacetic acid; 3-MFP, 3-0-methylfluorescein phosphate; p-NPP, p-nitrophenyl phosphate; PMSF, phenylmethylsulfonyl fluoride.

1

0003-9861/88 $3.00 Copyright All rights

Q 1988 by Academic Press, Inc. of reproduction in any form reserved.

HORGAN MATERIALS

AND

AND

METHODS

Transverse tubule preparation. Junctional elements of the t-tubules were prepared by the dissociation of heavy SR during centrifugation in ion-free sucrose gradients (7). The crude t-tubules were further purified by removing contaminating SR by the calcium phosphate loading procedure of Bonnet et al. (8), as modified by Hidalgo et al. (5). SR preparation. Fraction U2 was prepared from crude SR (SOOO-28,000 g preparation) by sucrose gradient centrifugation, as described previously (7). ATPase assay. Mg’+-ATPase (EC 3.6.1.3), (Ca’+, Mg’+)-ATPase (EC 3.6.1.38), and (Na+,K+)-ATPase (EC 3.6.1.37) were assayed as described previously (7). Activities in the presence of calcium alone were measured by the Pi release method in the presence of 8 mM Ca” and 5 mM ATP (5). Acetylcholinesterase (EC 3.1.1.7) activity. This was measured at 37°C in the absence and presence of 0.02% Triton X-100 using the procedure of Ellman et al. (9). Cholesterol. The total cholesterol content (cholesterol plus cholesterol esters) of the t-tubules was measured by the method of Allain et al. (10) using a cholesterol assay kit (Abbott Laboratories). Routinely, 50-100 pg of protein was added to 1 ml of reagent. Turbidity was corrected for by comparing absorbances at 500 and 640 nm, as described by Rosemblatt et al. (3). N&en&pine binding. Binding studies with [3H]nitrendipine (Amersham) were carried out at 25°C in pH 7.5, according to the method of 50 mM Tris-HCl, Fosset et al. (11). Nonspecific binding was measured in the presence of 1 pM nifedipine (Sigma). The maximum number of binding sites (B,,,) was determined from Scatchard plots. SDS-polyacqlamide gel electropharesis. The protein composition of the t-tubules was determined by electrophoresis in 10% gel slabs prepared and run by the method of Ames (12) which employs the buffer system of Laemmli (13). The gels were stained with 0.25% Coomassie brilliant blue and, after destaining were scanned with a Kipp and Zonen densitometer. Molecular weights were determined from a plot of log molecular weight versus mobility, calibrated with a 20,000-340,000 molecular weight kit (Boehringer). Phosphorylation experiments. Phosphorylation of t-tubule proteins (200 pg) was carried out in 0.5 ml of a basic incubation medium consisting of 20 mM histidine, 1 mM EGTA, pH 7.5. Other additions to the basic medium were as described in the figure and table legends. The reaction at 0°C was started by the addition of 0.9 pM [32P]ATP (60 Ci/mmol) (Amersham) and terminated after 10 s by the addition of 0.5 ml 10% TCA. The TCA-precipitated protein was washed three times with a solution containing 5%

KUYPERS TCA, 0.1 mM ATP, 1 mM phosphate, and finally once with ice-cold distilled water. The amount of 32P in the washed pellets was determined by Cerenkov counting. Immediately following counting, the pellets were dissolved in 1% SDS, 1% mercaptoethanol, 50 mM sodium phosphate (pH 2.4) and subjected to SDS-gel electrophoresis at pH 2.4 and 15°C as described by Avruch and Fairbanks (14). Following electrophoresis, one of the duplicate pair of gels was sliced into 1.5-mm portions and counted for “P content. The other gel was stained, destained, and scanned as described above. Detergent extraction of t-tubules. Lysolecithin extraction of t-tubule membrane proteins was performed as described by Hidalgo et a2. (5). Extraction with SDS was carried out by incubating the t-tubules for 20 min at 25°C in a medium consisting of 50 mM Tris-HCl, 3 mM ATP, 2 mM EDTA (pH 7.5) at a protein to SDS ratio of 2.5:1. The incubation mixture was then centrifuged for 30 min at 150,OOOg(5°C). The resulting pellet was then washed with 20 mM Trismaleate, pH 7.0, and recentrifuged for 30 min at 150,OOOg.The final pellet was resuspended in 0.3 M sucrose, 20 mM Tris-maleate pH 7.0. Estimation of membrane sidedness. The criteria and conditions used to estimate membrane sidedness of skeletal muscle plasma membrane (15) were applied to t-tubules, except that the acetylcholinesterase activity was unmasked with Triton X-100 instead of SDS. 3-0-Methyljluorescein phosphatase and p-nitrophenyl phosphatase activities. These were measured at 35°C using the assays described by Brandt et al. (16). Protein concentrations. These were measured using the method of Lowry et al. (17). RESULTS

Biochemical

characteristics

of t-tubules.

A comparison of the properties of t-tubules isolated directly from the sucrose density gradients following overnight centrifugation (crude t-tubules) with those of t-tubules which had contaminating SR removed by calcium phosphate loading (purified t-tubules) is shown in Table I. The calcium phosphate loading procedures resulted in a yield of 67% of the protein (in two fractions) and of that protein approximately 60% was in the fraction that did not load calcium, i.e., the t-tubules (mean of three experiments). The yield of purified t-tubules is 3.3 mg per 100 g of muscle. The reduction in the (Ca’+,Mg’+)-ATPase activity (Table I) shows that approxi-

TRANSVERSE

TUBULES

FROM TABLE

PROPERTIES

SKELETAL

MUSCLE

I

OF CRUDE AND PURIFIED

TRANSVERSE

Crude t-tubules” Yield (mg protein/100 g of skeletal muscle) Nitrendipine binding (pmol/mg protein) Cholesterol (pg/mg protein) Mga+-ATPase (gmol/mg protein/min) (Caa+,Me)-ATPase (Fmol/mg protein/min) (Na+,K+)ATPase (pmol/mg (protein/h) Acetylcholinsterase (Fmol/mg protein/min)

TRIADS

8.64 68.7 134 0.430 1.380 11.10 50.0

f 0.85 f 4.1 -+26 f 0.038 + 0.370 * 1.68 + 5.0

(lo)* (3) (5) (9) (6) (9) (5)

TUBULES

Purified

t-tubules*

3.30 95.5 226 0.614 0.164 18.72 77.0

0.35 19.1 26 0.059 0.050 3.18 12.0

f * f f k + k

(10) (4) (5) (9) (6) (9) (3)

a Crude and purified t-tubules refer to transverse tubules obtained directly from sucrose density gradients and those obtained after purification by the calcium loading technique of Hidalgo et al. (5). * Results are expressed as means * SE for number of observations in parentheses.

mately 90% of the contaminating SR was mately 70% of the protein in the purified removed by the loading procedure. We t-tubules. It is this preparation which is have determined the specific activity of used in all subsequent experiments. Detergent extraction. The lysolecithin the (Ca2+,Mg2+)-ATPase of purified SR to be 4.45 pmol/min/mg protein (7). Thus the extraction technique used by Hidalgo et al. level of SR contamination in the purified (5) was employed with our t-tubule prepat-tubules is 3.7% compared with 31% in ration in an attempt to purify and identify the crude t-tubules. the Mg2+-ATPase. The results (Table II In contrast to the (Ca2+,Mg2+)-ATPase and Fig. 2) agree with those of Hidalgo et activity, which shows a decrease, all the other properties listed in Table I show a 40-60% increase following purification. While cholesterol content, acetylcholinesterase activity, and (Na+,K+)-ATPase activity are markers for membranes of sur,104kDe face origin, nitrendipine binding sites occur primarily in the t-tubules of skeletal muscle (18). Our value of 95 pmol/mg protein compares with the values reported for t-tubules prepared by mechanical disruption of triads (85 pmol/mg) (19) and for the free t-tubule preparation of Rosemblatt et al. (3) of 56 pmol/mg (11). u Protein composition. The protein compositions of crude and purified t-tubules q I ,104kDe as determined by SDS-gel electrophoresis 70kDE / 30kD.m are shown in Fig. 1 as densitometer scans / of the gel patterns. In the crude t-tubules preparation, the major protein band (60% of the total) occurs at 104,000 Da while smaller peaks are at ‘70 and 30 kDa. Following purification of the crude t-tubule Migration e preparation by calcium loading, the 104kDa peak is halved in size while the 70FIG. 1. Densitometric scans of SDS-polyacryland 30-kDa peak are doubled. Together, amide electrophoresis gel patterns. (A) Crude t-tubules; (B) purified t-tubules. these three peaks account for approxi-

1

HORGAN

AND

KUYPERS

TABLE

II

EXTRACTION OF TRANSVERSE TUBULES WITH LY~OLECITHIN Protein distribution Protein Fraction

(%)

MgP+-ATPase (pmol/min/mg)

Cholesterol content (pg/mg protein)

Transverse tubules Supernatant 1 Supernatant 2 Pellet

100 44.4 + 4.6 (4) 19.2 + 2.0 (4) 37.5 z!z3.2 (3)

0.489 2 0.090 (4)’ 0.289 f 0.053 (4) 1.128 f 0.169 (4) 1.28 (1)

241 f 12 (3) 66 f 15 (3) 456 f 46 (3) 440 (1)

R?CO”Wy

104 kDa

70 kDa (7%of total protein)

30 kDa

24.1 f 1.9 (4) 14.6 + 4.7 (4) 16.2 + 2.8 (4) 7.80 (1)

17.7 f 1.6 (4) 44.0 k 5.6 (4) 15.1 + 3.5 (4) 6.8 (1)

17.1 + 1.1 (4) 8.21 + 0.8 (4) 31.4 f 4.4 (4) 30.1 (1)

0 Results are expressed as means f SE for number of observations in parentheses.

aZ. (5) in that most of the Mg’+-ATPase activity remained in the insoluble fraction following the first extraction. Following a second extraction with lysolecithin the soluble fraction had two to three times the Mg’+-ATPase activity of the initial t-tubules (Table II). Also, like Hidalgo et ab (5), we found that cholesterol copurified with the Mg’+-ATPase activity (Table II). Densitometer scans of the SDS-polyacrylamide gel electrophoresis patterns of the lysolecithin extracts of a t-tubule preparation are shown in Fig. 2. In this experiment a particularly clean separation of the various peaks was obtained. In

/

Table II the average peak areas from four experiments are shown. By comparing the gel pattern of t-tubule membranes (Fig. 2A) with that of the first soluble fraction (Fig. 2B) it can be seen that the 70-kDa protein is enriched in this soluble fraction. From the peak areas (Table II) this enrichment has been calculated as 2.5-fold. This suggests that the 70-kDa protein is easily removed by mild detergent treatment and therefore is probably an extrinsic membrane protein. The gel patterns of the second soluble fraction (Fig. 2C) and the insoluble fraction following two lysolecithin extractions (Fig. 2D) show enrich-

30kDa

/

30kDe

I

I Migrnnttion

e

FIG. 2. Densitometric scans of SDS-polyacrylamide electrophoresis gel patterns of lysolecithin extracted t-tubules. (A) Untreated t-tubules; (B) 1st supernatant; (C) 2nd supernatant; (D) insoluble fraction.

TRANSVERSE

TUBULES

FROM

ment of the 30-kDa protein. From Table II, which shows the protein distributions calculated from the peak areas, it appears that a good correlation can be obtained between the amount of the 30-kDa protein and the Mg”-ATPase activity of each fraction. Thus the overall conclusion to be drawn from the results in Table II and Fig. 2 is that the Mg’+-ATPase activity is associated with the SO-kDa protein. When t-tubules were extracted with SDS (Table III), the Mg’+-ATPase remained in the insoluble fraction as did the (Na’,K+)-ATPase activity. Figure 3 shows a densitometric scan of the SDS-polyacrylamide gel electrophoresis pattern of the insoluble fraction following the mild SDS extraction. The 70-kDa protein was virtually absent, again confirming its ease of removal by detergents. On the other hand, the 104- and 30-kDa proteins are concentrated and appear to be integral proteins of the t-tubule membrane. Phosphorylation experiments. In order to identify which of the t-tubule membrane proteins were associated with the (Na+,K+)-ATPase activity, SDS extracted t-tubules were incubated with [y-32P]ATP in the presence of various cations. The results (Table IV) show that a phosphorylated intermediate of the (Na+,K’)-ATPase is formed in the presence of Nat ions, and this formation is prevented by the presence of ouabain or K+ ions. The densitometric scan of the SDS-gel electrophoresis pattern (run at pH 2.4) of the t-tubules phorphorylated in the presence of Na+ and Mg2+ ions is shown in Fig. 4. Superimposed on this pattern is the distribution of 32P determined from Cerenkov

SKELETAL

/

t-tubules SDS-extracted t-tubules a Results

counting of slices from a duplicate gel. Under these conditions a considerable amount of protein did not enter the gel, presumably, as a result of the low pH. However very little 32P was associated with this high molecular mass fraction. It is evident that the 104-kDa protein was the only protein phosphorylated under these conditions and therefore would appear to be the (Na+,K+)-ATPase. Membrane sidedness. We used the method of Seiler and Fleischer (15) to estimate the sidedness of our t-tubule membrane preparations. The method assumes that acetylcholinesterase is located only on the outer face of the membrane and is therefore exposed in right-side-out membrane vesicles, while the (Na’,K+)-ATPase activity cannot be measured in completely sealed membrane vesicles since the ATP and ouabain binding sites are on opposite membrane faces. In order to find the optimum conditions needed to unmask latent III

activities

(Ca'+,M&ATPase

Protein

(Na+,K+)-ATPase

(fimol/min/mg)

(~mol/min/mg)

0.430 + 0.056 (4)

0.143 + 0.057 (4)

0.375

0.432 k 0.076 (2)

0

as means

+ SE for number

,3OkDa

FIG. 3. Densitometric scan of SDS-polyacrylamide electrophoresis gel pattern of t-tubules following extraction with SDS for 20 min at 25°C with a protein to SDS ratio of 2.51.

(fimol/min/mg)

are expressed

104kPa

COMPOSITIONOFSDS-EXTRACTEDTRANSVERSETUBULES

Enzymatic

M$t-ATPase

5

TRIADS

I

TABLE ENZYMATICACTIVITIESANDPROTEIN

MUSCLE

of observations

104

kDa

70 kDa (5% of total

2 0.110 (3)

22.7 + 1.6 (4)

0.449 * 0.064 (3)

27.9 zk 4.6 (3)

in parentheses.

composition

16.3

+ 1.2 (4) 0

30

kDa

protein)

17.1 + 1.8 (4) 28.2 + 4.9 (2)

6

HORGAN

AND TABLE

EFFECTS OF CATIONS ON [32P]P~~~~~~~~ [3zP]Phosphate (pmol/mg

Mg2

incorporation protein) 1 mM ouabain

12.40 52.30 16.08 13.66

MgZf, Na’ Mg2f, Na+, K+ Mg2f, Caa+

ND* 24.12 14.82 ND

a Concentrations of cations added as for Mga+-ATPase, *Not determined.

(Na+,Ka+)-ATPase activity, t-tubules were preincubated with various concentrations of SDS before the substrate was added (Fig. 5). The optimal SDS concentration was found to be 0.3 mg/ml for a t-tubule protein concentration of 1 mg/ml. The percentage latency of the (Na+,Ka+)ATPase, and thus the percentage of sealed vesicles, was estimated to be approximately 73% (Table V). Of the total acetylcholinesterase activity approximately 31% (Table V) was measured in the absence of detergent (Triton X-100) and this was taken as a measure of the right-sideMiirmtion

IV

INCORPORATION IN PURIFIED TRANSVERSE TUBULES

No ouabain

Additions”

KUYPERS

(Na+,K+)-ATPase,

ATP hydrolysis (rmol/min/mg protein) 0.562 ND 0.943 0.486 and (Caa+,Me)-ATPase

assay.

out plus leaky membranes. Thus when the percentage of leaky membranes, as determined by SDS unmasking of the (Na+,K+)-ATPase activity, was substracted, a value of 95% was obtained for t-tubule membranes sealed inside out. Hidalgo et al. (20) found that their preparations of t-tubules were predominantly sealed in the inside-out orientation, as were the junctional t-tubules prepared by mechanical disruption of triads (21).

-

I

7

I

4~.. ; :I II

/

30kDa

3 ._

: I l ...**I ?I. ...*. . .‘. ,. l **,** . . ., . . 5 Qel

15 Slice Number

25

FIG. 4. Densitometric scan of gel pattern obtained from SDS-polyacrylamide electrophoresis at pH 2.4 of SDS-extracted t-tubules phosphorylated by [““PIATP in the presence of Na+ and Mg2+. The dotted line shows the 32P content of slices from a duplicate gel.

FIG. 5. Effect of SDS on (Na+,K+)-ATPase activity. Transverse tubules at a protein concentration of 1 mg/ml were preineubated with various concentrations of SDS for 30 min at 25’C before the substrate was added and activity was measured.

TRANSVERSE

TUBULES

FROM TABLE

SKELETAL

MUSCLE

TRIADS

7

V

MEMBRANESIDENESSOF PURIFIED TRANSVERSETUBULES Activity No detergent

+ detergent

Latency

(RO i-L)

(L)”

23.53 + 5.56 (3)

76.53 f 11.77 (3)

69.3

30.8

-

Assay Acetylcholinsterase (nmol/min/mg protein)

Sidedness (% )

Ouabain-sensitive (Na+,K+)ATPase (rmol/h/mg protein)

4.00 f 0.68 (3)

14.68 f 2.81 (3)

72.8

(IO)*

(RO)’

95

5.0

27.2

a Leaky. *Inside-out. ’ Right-side-out.

Phosphatase activity. In Table VI, the rates of hydrolysis of ATP, 3-O-methylfluorescein phosphate (S-MFP), and p-nitrophenyl phosphate (p-NPP) by SR and t-tubules are compared. With all three substrates the SR ATPase activity in the presence of 5 mM Mg2+ is stimulated many fold by the addition of micromolar Ca’+. The largest stimulation was approximately 400-fold when 3-MFP was the substrate. In contrast, the t-tubular ATPase showed only slight stimulation by micromolar Ca2+.In some preparations no stimulation was observed, indicating that any observed stimulation was probably due to contaminating SR in the t-tubule preparation. In the presence of millimolar calcium alone, the t-tubules have up to six times the activity observed in the SR, showing that this activity is enriched in the t-tubule membrane. With ATP as the substrate, the t-tubular enzyme has approximately twice the activity in the presence of millimolar Ca2+as it has in the presence of Mg2+ alone. This confirms other reports (5) that the t-tubular ATPase is a (Ca” or Mg2+)-ATPase. DISCUSSION

In this paper we have described the further purification and characterization of t-tubules prepared by the dissociation of intact triads during centrifugation in ion-

free sucrose gradients (7). In agreement with other workers in this field, we found that t-tubules are characterized by (i) a high concentration of voltage-dependent Ca2+-channels as determined by nitrendipine binding sites (3, 11, 19, 22); (ii) a very high cholesterol content (3,23); (iii) a high specific activity of (Ca2+or Mg2+)-ATPase activity (3-5, 24); and (iv) a (Na+,K’)ATPase activity (4, 20, 21, 24). Most of the recent work with “free” ttubules has been carried out by two groups, namely Hidalgo and co-workers (3, 5, 20, 22) and Sabbadini and co-workers (24-26). Until now the only preparation of “junctional” t-tubules that could be compared with the free t-tubules were those of Caswell and co-workers (2, 4, 6,16,19,23). As reported previously our method of preparation yields t-tubules with properties almost identical to those of Caswell’s group (2, 4, 23). The main difference between the two types of t-tubules is that the a2+ or Mg2+)-ATPase of free t-tubules I; 24) is five times higher than that of ju’nctional t-tubules (4) (Table I). Since both Caswell and co-workers (21, 23) and we (Table V) have shown that most of the t-tubular vesicles prepared by dissociation of triads are intact, it is likely that the lower ( Ca2+ or Mg2+)-ATPase activity of junctional t-tubules is a real difference between the two types of t-tubules and is not due to damage to the membrane during preparation.

HORGAN

AND

KUYPERS

Detergent extraction of free t-tubules has been reported to result in enrichment of 107- and 30-kDa protein bands in one case (5) and 102- and 25kDa proteins in another (26). Our results (Tables II and III, Figs. 2 and 3) also show that proteins of molecular weights 104- and 30-kDa are integral proteins of junctional t-tubules. Okamoto et al. (26) have reported that many sarcoplasmic proteins are entrapped in t-tubule vesicles during preparation. This may explain the easy removal of the 70-kDa protein by mild detergent treatment observed in this study. Okamoto et al. (26) also tentatively identified the 102kDa protein as the Mg’+-ATPase in their free t-tubules. They also reported that the amount of 25-kDa protein in their preparations was variable and could be reduced by mild homogenization and the inclusion of protease inhibitors in their preparation. Our results, however, indicate that the 30kDa protein is the (Ca2+or Mg2+)-ATPase in our junctional t-tubules. No difference in the amount of 30 kDa protein was observed (results not shown) when PMSF was included in our preparation buffers. The results of Table IV and Figure 4 identify the 104-kDa protein in our junctional t-tubules as the (Na’,K’)-ATPase. This agrees with values reported for the cu-subunit of (Na+,K+)-ATPase from other tissues (27). However in all these studies a second subunit was also present. This psubunit had an M, of 38,000 in its unglycosylated form but when glycosylated behaved anomalously on SDS-polyacrylamide gels giving M, values ranging from 66,000 to 40,000 depending on the acrylamide concentration (28). In our studies the only major integral membrane protein in t-tubules besides the 104-kDa protein was the 30-kDa protein which appears to be the (Ca2+ or Mg’+)-ATPase (Table II, Fig. 2). Staining of SDS-polyacrylamide gels with “Stains-all” dye (29) showed that the 30-kDa protein was not a glycoprotein (results now shown) and therefore unlikely to be the P-subunit of the (Na+,K+)-ATPase. This interesting result raises the possibility that the (Na+,K+)ATPase present in t-tubules, which unlike previous sources of the enzyme are not

TRANSVERSE

TUBULES

FROM

surface membranes, may function with only the one subunit. In conclusion, the results presented in this paper support the hypothesis that the free and junctional t-tubules represent different functional elements of the t-tubular system analagous to the heavy and light elements of the SR (1). Positive identification of the integral membrane proteins of the junctional and free parts of the t-tubule is still required as is the elucidation of the roles of these two elements of the t-tubular system. REFERENCES

SKELETAL

MUSCLE

TRIADS

9

12. AMES, G. F. (1974) J. BioL Chem. 249,634-644. 13. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 14. AVRUCH,J.,ANDFAIRBANKS,G.(~~~~) Proc. NatL Acad. Sci. USA 69,1216-1220. 15. SEILER, S., AND FLEISCHER, S. (1982) J. BioL Chem., 257,13862-13871. 16. BRANDT,N.R.,CASWELL, A.M., AND BRUNSCHWIG, J. P. (1980) J. BioL Chem. 225, 6290-6298.

17. LOWRY,D. H.,RosEBRouGH,N.J., FARR, A.L., AND RANDALL,R.J. (1951)J. BioL Chem. 193, 265-275. 18. ALMERS,W.,FUIK,R.,ANDPALADE,P.T.(~~~~)J. Physiol. (London) 312,177-207. 19. BRANDT,N.R.,KAWAMOTO,R.H.,AND CASWELL, A. H. (1985) Biochem. Biophya Res. Commun. 127,205-212.

1. MARTONOSI, A. N. (1984) Physiol. Rev. 64, 1240-1320. 20. HIDALGO, C., PARRA, ~.,RIQUELME, G., AND JAI2. CASWELL,A. H., LAU, Y. H., AND BRUNSCHWIG, MOVICH,E. (1986) Bochim. Biophys. Acta 855, J. P. (1976) Arch. Biochem. Biophys. 176, 79-88. 417-430. 21. LAU, Y. H., CASWELL,A. M., GARCIA, M., AND 3. ROSEMBLATT,M.,HIDALGO,C.,VERGARA,C.,AND LETELLIER, L. (1979) J. Gen. Physiol. 74, IKEMOTO, N. (1981) J. BioL Chem. 256, 335-349. 8140-8148. 22. JAIMOVICH,E.,DONOSO, P.,LIBERoNA,J.L., AND 4. LAU, Y. H., CASWELL,A. M., AND BRUNSCHWIG, HIDALGO, C. (1986). Biochim. Biophys. Acta J. P. (19'77)J. BioL Chem. 252,556-5574. 855,89-98. 5. HIDALGO,~. A.,GONZALEZ,M.E.,AND LAGOSR. 23. LAU, Y. H., CASWELL,A. H., BR~JNSCHWIG,J.P., (1983) .I. BioL Chem. 258,13937-19345. BAERWALD, R. J., AND GARCIA, M. (1979) J. 6. BRANDT, M. R., CASWELL, A. H., AND BRUNBiol. Ch,em. 254,540-546. SCHWIG, J. P. (1979) Int. Congr. Biochem. 24. SABBADINI, R. A., AND OKAMOTO,V. R. (1983) A&r.,

454.

7. HoRGAN,D.J., AND KUYPERSR.(1987) ArchBiu them. Biophys. 253,377-387.

8. BONNET, J. P., GALANTE, M., BRETHES, P., DEDIEIJ, J. C., AND CHEVALLIER, J. (1978) Arch. B&hem.

Biophys.

191,32-41.

9. ELLMAN, G. L., COURTNEY,K. D., ANDRES, V., AND FEATHERSTONE,R. M. (1961) Biochem. Pharmacol. 7,88-95. 10. ALLAIN, C. C., PooN,L., CHAN, S. G.,RICHMOND, W., AND Fu, P. (1979) Clin. Chem 20,470-475. 11. FOSSET, M., JAINOVICH, E., DELPONT, E., AND LAZDUNSKI, M. (1982) J. Biol. Chem 258, 6086-6092.

Arch. Biochem. Biophys. 233,107-119.

25. SCALES,D.J.,AND SABBADINI,R.A.(~~~~)J. Cell. BioL 83,33-46.

26. OKAMOTO,~. R.,MOULTON, M. P.,RuNTE,E. M., KENT, C. D., LEBHERZ, H. G., PAHMS, S. A., AND SABBADINI, R. A. (1985) Arch. Biochem. Biophys. 237.43-54.

27. JORGENSEN,P. L. (1982) Biochim. Bioph,ys. Actu 694,27-68. 28. PETERSEN,G.L., AND HoKIN,L.E.(~~~~)J. BioL Chem. 256,3751-3761.

29. KING,L. E., AND MORRISON,M. (1976)Anal. Bicthem. 7 1.223-230.