313 PROPERTIES OF THE SARCOLEMMA*

C. Y. MATSUSHWA California State Polytechnic University, Pomona

Like other surface membranes, the sarcolem of cells serves to maintain the integrity of the and to regulate the constant exchange of ions and metabolites between the inter- and intracellular environments. Its vital role in excitation-contraction coupling is well documented, but its role in the conversion of muscle to meat remains unclear. Advances in membranology indicate that the chemical constituents are intimately associated with structure and function of membranes. Thus, knowledge of the chemical and structural characteristics of the sarcolemma would greatly increase our compre- hension of such phenomena as bioelectrogenesis, metabolite transport and permeability changes in this surface membrane. Since Dr. Stromer has provided us with an insight into the structure of membranes, I will limit my discussion to some chemical properties of skeletal muscle sarcolemma.

As a prelude to describing these properties, some time should be devoted to discussing available isolation procedures. "his is necessary because the chemical characteristics of sarcolemmal preparations appear to be influenced by the isolation procedure utilized (Abood -et ,',a1 1966; Heffron et al., 1967; Hultin and Westort, 1969; Sulakhe Let a1-*, 1973a). Westort and Hultin (1966) also inferred that the procedure of choice may be governed by the objectives of the study.

Isolation of Sarcolemmal Membranes

Since 1961, numerous isolation procedures for skeletal muscle sarcolemma have been published (Abood et al., 1966; Boegman et al., 1970; Koketsu et al., 1964, Kono and Colowick, 1961; McCollester, 1962; McNamara et a1 ., 1971; Peter, 1970; Rosenthal et al ., 1965; Westort and iIultin, 136r These procedures share the common objective of solubilizing and extracting the myofibrillar proteins without membrane disruption. Furthermore, these procedures usually represented modifications to those originally reported by Kono and Colowick (1961) and McCollester (1962) who utilized high and low ionic strength extracting solutions, respectively, and hence, sarcolemmal isolation procedures can be classified as being either high ionic strength or low ionic strength extraction methods.

* Presented at the 27th Annual Reciprocal Meat Conference of the American Meat Science Association, 1974. 314 The basic scheme to Kono and Colowick's procedure (Kono and Colowick, 1961) is shown in Figure 1. Essentially, this procedure entails repeated homogenization in Tris buffer and the myofibrillar proteins extracted with 0.4 M LiBr followed by overnight extraction in 1.0 M KC1. Final separation of the fragments from extraneous material is accomplished by differential centrifugation through a series of KBr solution differing in density. The major modifications to this method have been to homogenize in buffered 50 mM CaC12, to centrifuge at lower gravitation forces, i.e., 900 to 2000 x g, and eliminate the differential centrifugation through KBr (Abood et al., 1966; Koketsu et e.,1964; Sulakhe et al., 1973a). According to Koketsu --et al. (1964r the use of CaC12 during homogenization was necessary to prevent tangling and destruction as well as adhesion of the membrane fragments to the container during isolation. The elimination of differential centrifugation through KBr was facilitated by utilizing lower centrifugal forces which separates the sarcolemmal fragments from particulate matter (Sulakhe et al., 1973a).

Because strong salt solutions may have adverse effects on membranes, McCollester (1962) developed a milder procedure for preparing sarcolemmal membranes . This procedure maintained near physiological conditions by employing a maximum ionic strength of 0.15 M and pH values between 6.4 and 7.8. The rationale to this procedure, however, is difficult to reconcile. As shown in figure 2, was repeatedly homogenized in 50 mM CaC12 to prevent irreversible contraction and/or activate Ca2+- dependent enzymes during the subsequent incubation step. Hultin and coworkers (Hultin and Westort, l969a; Stanley and Hultin, 196%), however, indicated that the primary effect of CaC was to stimulated ATPase activity and, thereby destroying a'it 1 endogenous ATP and permitting actomyosin to form. At any rate, the sediment following centrifugation was washed 4 times in a buffered NaCl solution before the cell segments were incubated at 37OC for 30 minutes. According to McCollester (McCollester, 1962; McCollester and Semente, lw),incubation was necessary to break down the 'lcytoskeletonn of the and thereby transforming the contractile proteins from a water-insoluble to a water-soluble state. He suggested the "cytoskeleton" consisted primarily of the and Z lines. With respect to the latter, a recent phase micro- scopic study indicated that the Z lines were degraded during the incubation step (Matsushima, 1971) . Following incubation and 5 post-incubation washes with histidine-buffered 25 mM NaC1, the muscle cell segments were subjected to several Tris-buffered water extractions. During these water extractions, the contractile proteins were dissolved, and the cell segments were emptied leaving the sarcolemmal membranes. The emptying process was characterized initially by marked swelling and a concomitant increase in viscosity of the myofibrillar proteins; this was followed by a subsequent decrease in both viscosity and volume of the sedimented material containing the sarcolemmal membranes as dissolution of the contractile proteins occurred. Several alternatives have been reported for the dissolution of the acto- myosin gel and liberation of empty cell segments. These include: (1) first extracting with 2 x 10-7 N NaCH md then treating the residue with ATP (Rosenthal et al., 1965) or sodium pyrophosphate (Ferdman et al., 1970) and waterT(3 first extracting with 0.01 mM EGTA followed by FIGUFG 1

ISOLATION OF SKELETAL MUSCLE SARCOLEMMA USING HIGH IONIC STRXNGTH SOLUTIONS (Kono and Colowick, 1961)

(a) Suspend in 0.4 M LiBr, 0.01 M Tris (pH 8.2-8.4) (b) Stir 4 hr. (c) Centrifuge at 20,000 x g for 25 min .

(a) Repeat previous steps except stir for 3 hr. FIGURF: 2

ISOLATION OF SKELETAL MUSCLE SARCOLEMMA USING LQI*J IONIC STREDGTH SOWIONS (McCollester, 1962)

Muscle Tissue

(a) Homogenized in 50 mM CaCb for 10 sec. (b) Homogenate filtered through cheesecloth c) Centrifuge at 600 x g, 5-7 sec.

Sediment

(a) 4 washes in 25 mM NaC1, 2.5 mM DL-histidine chloride (pH 7.4) (b) Centrifuge at 600 x g, 5-7 see. between washes

Supernatant Sediment

Suspend in the NaC1-histidine solution Incubate at 370C for 30 min. Centrifuge at 600 x g, 5-7 sec. I Supernatant Sediment

(a) 5 washes in the NaC1-histidine solution (b) Centrifuge at 600 x g, 5-7 sec . after each wash.

Super atant

(a) 4-5 "extractions" with Tris-buffered water (PH 7.4-7.8) Centrifuge at 2000 x g, 7 min., after each (b) "extractnt ion .I1 Supernatantrt Sediment = empty cell sewents using 0.1 mM ATP to remve any residual actomyosin (Peter, 1970), or (3) direct application of the Tris-water extract on a double layer of sucrose (21% over 6036 sucrose) and centrifugation at 900 x g (Westort and Hultin, 1966).

Regardless of the procedure utilized, the final preparation contains empty cell segments which appear through phase optics as tubular and transparent structures (Figure 3 ) . Occasionally, cable-like elements have been observed coiled around the external surface of these empty tubular segments (Kono and Colowick, 1961; Matsushima, 1971). At the resolution attainable by the electron microscope, the sarcolemma (Figure 4) a pears as electron-dense ribbons, approximately 80 8 wide (Matsushima, 1971P , with a mat of fine fibrils adhering to what probably represents the external surface of the membrane. Several authors (Kono and Colowick, 1961; Kono et al., 1964; Hultin and Westort, 1969b; Rosenthal et al., 1965) have suggested that these fibrils represent the outer layer of fibrous often associated with the term, sarcolem (Mauro and Adams, 1961; Robertson, 1956). On the other hand, McCollester (1962) is of the opinion that these fibrils are contaminants which may account for up to one-half of the dry weight of sarcolemmal preparations.

Purity of Sarcolemmal Preparations

The assessmerit of purity of sarcolemnal preparations has been very difficult to ascertain. One major obstacle is the lack of any effective marker for this membrane. As a result, most investigators have had to rely on microscopic, enzymic markers for and known properties of sub- cellular organelles, and/or exhaustive comparative studies to determine purity of their preparations. An example utilizing enzyme markers is shown in table 1. In accord with other studies utilizing electron microscopy and/or enzyme markers such as succinic dehydrogenase (Matsushima, 1971) or succinic oxidase (Heffron and Duman, 1967; Hultin and Westort, 1969b), contamination from mitochondrial membranes appears minimal at best. The presence of microsomal membranes in sarcolemmal preparations, on the other hand, has been difficult to assess. If glucose-6-phosphatase activity, which is commonly used as a marker for microsomes, is utilized, it would appear that membranes of the sarcotubular system are present in significant quantities. However, as I will show and point out later, Hultin and Westort (l969b), who observed similar activities in their sarcolemmal preparations, have presented suggestive evidence to the contrary. Sbnilarly, exhaustive comparative studies by Sulakhe 5 al. (1973a,b) suggest minimal contamination by sarcoplasmic reticular membranes .

Still more difficult has been the assessment of contamination by the myofibrillar proteins. To date, no quantitative method of determining the presence of these proteins in sarcolemmal preparations have been published. However, qualitative methods have been reported (Madeira and Carvalho, 1972; Matsushima, 1971; Peter, 1970). -0 S S 0 v-

aL

c 319

Figure 4. Electron micrograph of isolated sarcolemmal membranes. X90,720. TABLE 1. COIQARISON OF SUCCINIC DMYDROGZNASE AND GiXCOSE-6-PHOSPKATASE ACTIVITIES IN MITOCHONDRIAL, MICROSOMAL, AND SARCO~LFRACTIONS"

Fraction Succinic Dehydrogenaseb Glucose -6 -Phosphatasec

Mitochondria 99.33k14.43 4.43k1.79 Microsomal 27.0125.46 7.OYLl.43

Sarcolemma o.6mo.33 1.&to .83 a Taken from Matsushiraa (1971). Specific activity expressed sts males DCIP reduced per min. per mg proteinkstandard error at 38OC. C Specific activity expressed as nmoles Pi liberated per min. per mg proteinkstandard error at 30°C.

Chemical Properties of the Sarcolemma

With isolation procedures readily available, numerous studies have been conducted to determine the composition and associated properties of the sarcolemma. The extensive microscopic and chemical analysis of rat and bullfrog skeletal muscle sarcolemma by Kono and associates (Kono and Colwick, 1961; Kono et al., 1964) and Abood et al. (1966), respectively, provided the first compositional data. As shown in Table 2, the sarco- lemma, like other membranes, are composed largely of protein (approximately 6746) and lipids (approximately 16$) and to a much lesser extent poly- saccharides, nucleotides, protein phosphorous, and ash. Although Kono and Colowick (1961) reported similar values, Kono et al. (1964) later observed that lipid content of the rat skeletal muscle sarcolemma may be as high as 23$ on a dry weight basis, and that of the 23% approximately 33$ was phospholipids and about 52% was triglycerides and cholesterol. Interestingly, a survey of the literature will show a 1000 fold variation in phospholipid content of sarcolemmal preparations when expressed as phospholipid phosphorous per mg protein (Table 3). Part of the variation in total lipid and phospholipid content among membrane preparations my be due to the relatively small quantitative contribution the plasmalemma makes to the entire three layered structure surrounding the muscle cell. Kono et-- al. (1964) estimated that the plasmalemma contains about 30$ of the total protein when the sarcolemma is defined as a three layered structure surrounding the sarcoplasm. Hence, the presence of the outer fibrous connective tissue and intermediate amorphous layer in sarcolemmal preparations would greatly dilute the phospholipid content of the inner- most plasmalemma. A second possible explanation to the variation in reported phospholipid content may be attributed to variations in degree of contamination by intracellular membranes or non-membraneous elements. TABU2 2. COMPOSITION OF BULLFROG SARCOLEMMA'

~~ -

Material $ dry weight

Protein (~owry) 67.&4 .O Lip id 16 .e1.5 Polysaccharide o.po.2 Nucleotides 1.550.4 Protein phosphorous 0.2 Total ash 1.8~0.5 a Taken from Abood et al. (1966)

TABU 3. PHOSPHOLIPID PHOSPHOROUS VALUES FOR FOR SARC0L;EMMAL MEMBRAJ!JrES OF SKELETAL MUSCL;Ea

ug phospholipid Mus cle Reference ~/mgprotein source

Boegman et al. (1970) 1430 frog Ashworth and Green (1966) 11-12 rat Kono and Colowick (1961) 12 05 rat Abood --et al. 3 -8 frog (1966) rat &no et al. (1964) 1-35 Hultin and Westort (1969) 1.1 chicken

a Except for values reported by Boegman et al. (1970), phospholipid values were calculated by Hultin and Westort (1969). Recently, Fiehn c-et al. (1971) compared the lipid content of sarco- lemmal, fragmented sarcoplasmic reticular (FSR) and mitochondrial membranes (Table 4). Both FSR and mitochondrial membranes were found to be rich in phospholipids whereas the ratio of phospholipids to neutral lipids was approximately 1 in sarcolemmal membranes. Detailed analysis of the neutral lipids (table 5) showed the major component of both subcellular membranes was triglycerides, whereas cholesterol represented the major neutral lipid in the sarcolemma. Esterfied cholesterol represented about 3076, 16 and 5% of the total cholesterol in sarcolemmal, FSR and mito- chondrial preparations, respectively. Interestingly, Martonosi ( 1972 ) reported that 90$ of the cholesterol in sarcoplasmic reticulum membranes are unesterified and that the mitochondria contains very small quantities of this lipid. Fiehn et-- al. (1971) also found significant quantities of free fatty acids in their sarcolemmal preparations but were unable to effectively explain this finding.

In a similar analysis of the phospholipid component (Tdble 6), all three membranes were found to be rich in phosphatidylcholine and phospha- tidylethanolamine; however, the sarcolemma possessed larger quantities of phosphatidylserine and sphingomylein than did the subcellular membranes. Other workers have reported small quantities of phosphatidylinosital (Abood et al., 1966; Kono and Colowick, 1961) and phosphatidic acid (Abood et a1., 1966) in their sarcolemmal preparations. Fiehn --et al. (1971) also noted a distinct difference in molar ratios of cholesterol to sphingomylin between the three membranes. Molar ratios of 1.6, 3 and 4.5 for mitochondrial, FSR and sarcolemmal membranes, respectively, were reported. The molar ratio of cholesterol to phospholipids calculated to 0.57 which agrees with those reported for rabbit muscle cell membranes by Severson --et al. (1972). The significance of such ratios is twofold and rather obvious. First, they probably reflect upon the structural and functional specificities of each membrane (Ashworth and Green, 1966; Fiehn et- a1-*, 1971; Martonosi, 1971). Secondly, such ratios may provide for a more critical evaluation in defining the origin of membranes in isolated preparations.

The problem of degree of contamination from both membraneous and nonmembraneous elements has been an obstacle in characterizing the proteins and enzymes associated with this surface membrane. As shown earlier (table 2), proteins represented abaut 67% of the sarcolemma. In conjunction with this, an amino acid composition of bullfrog sarco- lemmal proteins was reported by Abood et al. (1966) and compared to rat muscle membrane and calf skin collagenTtable 7). Their findings, as indicated by much smaller quantities of glycine and hydroxyproline, confirmed electron microscopic observations that connective tissue was almost completely removed during their final puilficatfcn with 0.6 M KC1. The differences between both bullfrog and rat muscle membranes when compared to would make it appear reasonable to consider the connective tissue elements as contaminants and that the term, sarcolemma, be restricted to at least the basement and plasma membranes if not the plasmalemma per se. On this premise, the magnitude of connective tissue contamination can be drawn from studies carried out by Kono --et al. (1964) 325

TABU 7. AIJIINO ACID COMPOSITION OF VARIOUS MEMBRANE PREPARATIONSa

Rat muscle Calf skin Amino acid Ib I1 I11 membranes collagen

Cystine 1.35 0 .3 0 *3 0 93 0 .o 0 .o Aspartat e 8.720.8 10.0 3 -0 5 -8 4 *5 Threonine 3 .g+-O.k 5 *7 2.2 2 *9 1.8 Serine 5 20.3 6.2 4.1 4.2 3 *7 Glutamate 7.720.5 10.7 4.2 7 08 7 *2 Proline 6.90.5 6.1 15.0 7 -8 13.6 Glycine 13.251.2 14.3 24.5 25.6 32.6 AlanSle 6.7k0.4 8.0 12.3 9 -8 11.1 Valine 2.4i0.1 5 00 2 05 3 98 2.2 Methionine 1.5t0.1 2.4 0.5 1.o 0.6 Isoleucine 3.90.4 4 03 1.o 2 *5 1.1 Leucine 5.350.4 6 83 3 *9 4.1 2 *5 Tyrosine 2.3k0.2 2.4 0.6 1.o 0 e3 Phenylalanine 2 .9~0.3 2.6 1.1 2.1 1*3 Histidine 2 .OrO.l 1-7 0.8 0 *9 0 -5 Lysine 6.3k0.5 6.8 5 03 3 *O 2 -7 Arginine 3 Jk0.3 5 03 2.1 4 ,O 5 00 Hydroxyproline 0 .7ko .2 2 .o 9 *o 6.2 8 05 a Taken from Abood et al. (1966). b I = summer frogs; I1 = winter frogs; I11 = 3%of membranes not hollow and transparent. All values residues/100 residues. who estimated that this component may represent as much as 37'$ of the dry weight of isolated membrane preparations. The second noteworthy observation was the relatively large quantities of the acidic amino acids, glutamate and aspartate, in the sarcolemma. It was suggested that these negatively charged amino acids may serve as additional binding sites for cations involved in regulating membrane permeability and electrical properties of this membrane.

The need for effective markers and the recognition of its functional roles in transport and cellular metabolism have stimulated much interest in enzymes associated with the sarcolemma. Table 8 shows some enzyme activities observed in isolated membrane preparations. Although the majority of these enzymic activities are consistent with those observed in plasma menibranes of other tissues, the presence of lactate dehydro- genase and, in particular, glucose -6-phosphatase activities raises several interesting questions. For instance, are these enzymes associated with the membrane structure in situ or redistributed during 327 homogenization (Hultin and Westort , 1969b; Warren and Glick, 1971)? Secondly, do these activities, in particular, glucose-6-phosphatase, signify sizeable contamination from sarcotubular membranes in isolate sarcDlemma1 preparations? Although these questions are difficult to answer, Hultin and Westort (1969b) presented suggestive evidence indi- cating glucose-6-phosphatase is associated with the surface membrane of skeletal muscle. One line of evidence was shown previously (table 3), i.e., a low phospholipid phosphorous content in their sarcolemmal preparation (1.1 ug P/mg protein). However, investigators observing glucose-6-phosphatase activities in their preparations have neglected to consider the T-tubules as a site for this enzyme. It is known the T-tubules are invaginations of the sarcolemma (Franzini-Armstrong and Porter, 1964) and are not in direct communication with the sarcoplasmic reticulum (Franzini-Armstrong, 1970). Since our knowledge of the properties of this system is vague (Martonosi, 1972), it is possible that the transverse elements remain attached to the surface membrane during isolation.

In a similar fashion, acetylcholinesterase activity is of interest. Histochemical studies (Namba and Grob, 1968; 1970) indicate that the motor endplate remains attached to the sarcolemma during isolation and that cholinesterase activity is localized almost entirely to this particular area in isolated membrane preparations. In addition, Nanba and Grob (1968) indicated that this enzyme is tightly bound to the membrane. The significance of these findings are chvious and requires no elaboration.

Because Na', K+ and Ca2+ are intimately involved with excitation- contraction coupling (Sandow, 1965), a number of studies have been directed toward identifying and characterizing the enzymes involved with the transport of these cations across the membrane. It is well documented that these enzymes are ATPases since movement of these cations is against an electrochemical gradient. Like plasma membranes from other sources, the ATPase of sarcolemma involved with Na+ efflux and K+ influx is activated by Mg2' alone, but requires the presence of Na+ and K+ for optimal activity. As shown in table 9, the Mg2+-dependent- (Na+ + @)-stimulated ATPase is sensitive to the cardiac glycoside, ouabain, as well as to oligonycin and diphenylhydantoin (DPH). The inhibition by DPH was of particular interest to Peter (1970), and by comparing ATPase activities of various subcellular components in the presence of these inhibitors (table lo), he observed that DPH specifically and completely inhibited (Na' + K+)-stitmlated ATPase activity of the sarcolemma. He concluded that sensitivity of the sarcolemmal enzyme to dephenylhydantoin may be a useful marker for sarcolemmal melllbranes. Others have shown that activity of this transport enzyme is also inhibited by F' ions (Stam et &., l969), by certain sulfhydryl reagents such as p-chloromercuribenzoate and N-ethylmalehide (Hotta and Usami, 1967; Matsui and Schwartz, 1966) and by certain pharmacological agents such as quindine (Samaha and Gergley, 1966) and strophanthin K (Stam et al., 1969). In addition, Skou (1965) pointed out that Ca2+ inhibits ATPase activity due to Na+ plus K+. Although several workers have TABU 8. ENZYME ACTIVITIES OBSERVED IN ISOLATED SARCOIZMMAL MEMBRANE PREPARATIONS

-

Enzyme Reference

Acetylcholinesterase Ferdman et al. (1970); Namba and Grobe (1969, 1970); Severson --et al. (7357. Adenylate cyclase Severson I-et al. (1972). AMP aminohydrolase Ferdman --et al. (1970).

( ) W ATPase s ril Ch

Ca2+ - stimulated Ferdman etI- al. (1970); McNamra L-et al. (1971); Peter (1970); Severson c-et al. (1970); Sulakhe --et al. (1973b). Mg2+ - stimulated Andrew and Appel (1973); Boegman et al. (1970); Matsushima (1971); (Na' + e)- stimulated - Mg2' Peter (1970); Sulakhe et-- al. (1973b); McNamara et-- al. (1971). dependent Glucose -6-Phosphosphatase Matsushima (1971); Hultin and Westort (1969). Lactate Dehydrogenase Hultin and Westort (1969). p-Nitrophenylphosphatase Sulakhe --et al. (1973). TABLE 9. CHARACTERISTICS OF THE ATPASE(S) OF SARCOLEMMAa

Prep 2+-ATI?aseb Mg2+ f saf + K') - ATPaseb no. YieldC ---( Dma Oligomycin Ouabain

1 0.185 ~.log 0.108 1.136 0.0gi ------

2 0.143 0.152 0.159 0.232 0.166 0.124 0.1% 3 o.icj6 0.1% 0.146 0.226 0.168 0.100 0.201 a Taken from Peter (1970). b Specific activity expressed as moles Pi liberated/min/mg protein. Expressed as ag sarcolemnal protein/gm muscle. d DPH = diphenylhydantoin.

TABU 10. SPECIFICITY OF DIPHENYLHYDANTOIN EFFECT ON ON SARC0L;ESIIMAL (Na' + K+)-ATPasea

Source of ATPaseb -Addit ions None DPH Ouabain Oligomycin

~~ ~~~~

Mitochondria 0.406 0.381 0.403 0.166

Fragmented sarcoplasmic reticulum 0.658 o .662 o .618 0.447

Myosin B 0 .lo2 0 .lo2 0 .lo2 ---

Sarcolemma 0.193 0.142 ------a Taken from Peter (1970). b All enzyme activities were measured in a medium employed for assay of (Mg2+ + Na+ + K+)-ATPase of sarcolanma. For exact assay conditions, see original paper. Specific activity expressed as moles Pi liberated/min/mg protein. 329

observed such an inhibition in cardiac sarcolemma (Stam et al., 1969; Sulakhe and Dhalla, 1971), Ca2+ inhibition of skeletal muscle sarco- lemmal ATPase activity has been difficult to demonstrate.

Recently, Sulakhe --et-al. (1973b) reported that Mg2+-ATPase activity was enhanced by low concentrations of Ca2+, and furthermore, ATP hydrolysis was maintained with Ca2+-ATP as substrate (table 11). Ca2+-stimulated ATPase activity in sarcolemmal membranes have also been reported by Andrews and Appel (1973), Peter (1970), Severson et al. (1972), Stam et e. (l969), Sulakhe and Dhalla (1971), and Sulakhe et-- al. (1973a). Table 11 also shows two lines of evidence used by Sulakhe --et al. (1973b) to demonstrate that the observed enzyme activities were a property of the sarcolemma and not FSR membranes, i .e ., (1) aU. three enzyme activities and' the magnitude of stimulation of Mgz+-ATPase activity by added Ca2+ were much less in sarcolemmal than FSR membranes, and (2) Ca-ATPase activity of sarcolemmalmembranes was greater than Mg-ATPase, while in the sarcoplasmic reticulum both activities were similar. Although several other lines of evidence were provided to show Caz+-ATPase activity was endogenous to the sarcolemma (Sulakhe and Dhalla, 1971; Sulakhe et al., 1973b), two uestions still remained. First, is ATP hydrolysis in the presence of &+or Ca2+ due to the same or two different enzymes? Secondly, what is the functional significance of Ca2+-stimulated ATPase activity in the sarcolemma?

With respect to the second question, A. Weber (1966) suggested that the muscle cell membrane possessed, in addition to the sarco- plasmic reticulum and mitochondria, a calcium transport system. The possibility of such as transport system in the cell membrane was deduced from two known facts. First, a large Ca2+ concentration gradient exists across the cell membrane, and secondly, the sarcolemma is permeable to calcium. In light of this, several investigators (Sulakhe and Dhalla, 1971; Sulakhe et al., 1973a,b) have speculated that the Ca2+-activated ATPase is involved with this transport system. Recently, Sulakhe and associates (Severson --et al., 1972; Sulakhe et al., 1973a) observed energy-linked calcium binding in their sarcolemmal preparations. As shown in table 12, calcium binding of sarcoleml membranes was substan- tially increased in the presence of ATP and further enhanced by added oxalate. Since slmilarities in ATP-dependent calcium binding were observed for both sarcolemmal and FSR membranes, these investigators carried out an experiment similar to that devised by Martonosi (1968) to demonstrate Ca2+ binding was an endogenous property of the surface membrane. In this experiment, minced rabbit muscle was incubated with phospholipase C before isolation and measurement of Ca2+ binding by the respective membranes. The results of this experiment are shown in table 13. Phospholipase C treatment cause a marked reduction in calcium binding and accumulating ability of the sarcolemma, while activities of FSR isolated from phospholipase C treated muscle were similar to those from control tissue. Similarly, both Mg2+-stimulated and Ca2+-stimulated ATPase activities of the sarcolemma were reduced in phospholipase C treated muscle, whereas the corresponding enzyme activities were unaltered in FSR isolated frcm the same muscle. It was also demonstrated that the 330

TABLE 11. EFFECT OF Mg2+ ANTI Ca2+ ON ATPase OF SKELETAL MUSCLE SARC0L;EMMA"

ATPaseb Activity Sarcolemma Sarcoplasmic Reticulum

Mg -ATPase 0.23 * 0.02 0.55 f 0.04 MgCa -ATPase 0.49 r 0.03 2.08 * 0.15

Ca-ATPase 0.38 f 0.03 0.54 * 0.03 a Taken from Salukhe et al. ( 1973b). Specific activity expressed as nmoles Pi liberated/mg protein/min.

TABU 12. CAICIUM BINDING AND ACCUMULATION ACTIVITIES OF SKEIXTAL MUSCLE SARCOLEMMA AND SARCOPLASMIC RETICULUMa

Calcium binding or accunnrla tionb Sarcolemma Sarcoplasmic reticulum

-ATP 3.10 k 1.80 6.80 k 0.95

+ATP 18.36 k 1.9 202.55 f 6.65 +ATP + oxalate 189.90 * 11.30 2427 .OO * 162 .OO a Taken from Sulakhe et al. (1973a). Activity expressed as nmoles Ca2+ bound or accumulated per mg protein per 30 sec. TABLE 13. CALCIUM BINDInTG AND ACCUMULATING ACTIVITY OF SAFC0I;EMMA AND SARCOPLASMIC RETICUWM ISOL&L"D FROM CONTROL AND PHOSPHOLIPASE C TRIUSED bWSCr;Ea

Calcium Bound or Accumulated b Sarcolemma Sarcoplasmic Reticulum- - Assay Loss of LOSS 01 conditions Control Treated activity Cclntrol Treated activity

+ATP

+RTP + oxalate 200 25 87.5% 2000 1900 5$ a Taken from Sillakhe etc- a1 .( 1973a). b Activity expressed as nmDles Ca2+ bound 3r accmdated per mg protein per 30 sec. 332 calcium binding system of sarcolemmal membranes was more labile and exhibited a more rigid requirement for ATP as a phosphate donor when compared to the sarcoplasmic reticulum. In a recent review, Martonosi (1972) pointed out that nucleoside triphosphates other than ATP, carbolrlylphosphate and p-nitrophenylphosphate may effectively serve as energy donors for Ca2+ transport in the sarcoplasmic reticulum. The findings of Sulakhe and associates (Severson et al., 1972; Sulakhe -et a1-*, 1973a,b) lend support to Weber's (Weber, 1966) contention that an active Ca2+ transport system is localized in muscle cell membranes.

It is also interesting to note that several investigators (Koketsu et al., 1964; Madeira and Carvalho, 1972) have observed ATP-independent binding of Ca2+ in isolated sarcolemmal preparations. In this case, H+ ions are released with calcium binding. Furthermore, most of the calcium appears to bind to the lipid fraction, probably to the phos holipids (Koketsu g.,lw), and at the same sites that bind Mg2' and certain local anesthetics such as quinine and tetracaine (Madeira and Carvalho, 1972 1 The role of Ca2+ in excitation-con*raction coupling and the contraction-relaxation cycle in muscle is well documented. From the preceding discussion, it appears that the sarcolemma may possess two separate Ca2+ binding systems or one system carrying out two functions. On one hand, ATP-independent Ca2+ binding may be associated with stabilizing and regulating the permeability and electrical activity of the sarcolemma (Koketsu _.-et al., 1964; Madeira and Carvalho, 1972). Such a role for calcium has been thoroughly reviewed (Manery, 1966; Rothstein, 1968). This type of binding appears to be an ion-exchange type of adsorption and desorption at negative sites on the membrane (Manery, 1966) since Ca2' binds at sites originally occupied by H+ ions (Madeira and Carvalho, 1972). On the other hand, the energy-linked Ca2+ binding observed by Sulakhe and associates (Severson, 1972; Sulakhe -et al,') 1973a,b) may be related to Ca2+ efflux during relaxation (Martonosi, 1972; Sulakhe and Dhalla, 1971). Martonosi (1972) indicated that active Ca2+ transport by the cell membrane may be the principle regulator of the contraction-relaation cycle in amphibian muscle. However, if we assume specific activity as shown in table 12, is indicative of trans- port ability, the sarcolemma would play a secondary role to the sarco- plasmic reticulum in regulating cytoplasmic Ca2+ concentrations in mammalian skeletal muscle.

Thus far, nry discussion has been limited ta muscle as a functional organ. With regard to post-mortem muscle, research on changes in the sarcolemma is limited. Studies to date, however, suggest the sarcolemma is very susceptible to physical damage. Dawson (1966) and Osner (1966) reported that the rate and amount of enzyme efflux is influenced by freezing and thawing as well as by the rate of freezing. Osner (1966) also observed increased membrane permeability to intracellular proteins when muscle was allowed to pass through rigor while attached to the carcass. That structural alterations of the sarcolemma do occur has been confirmed by Reed --et al. (1966) who microscopically studied pre- and post- 333 rigDr pork muscles. These investigators suggested that the surface membrane is very labile and subject to rapid changes afker the death of the animal. This appears plausible in view of Dawson's (Dawson, 1966) experimental finding. In this experiment, whole chick muscle was incubated in physiological saline solution at 37%, and after 1 hour, detectable intracellular marker enzyme activities were found in the surrounding incubation medium.

It is obvious that changes in membrane permeability characteristics is one phenomenon occurring in post-mortem muscle. This is undoubtedly involved with smokehouse and cooler shrink. At the Second Annual Meat Industry Research Conference, Stramer (1966) also eluded to an inthate involvement of post-mortem changes in sarcolemmal membranes with such phenmena as soft, watery pork and penetration and retention of curing ingredients. In addition, it was shown earlier that the plasma membrane possesses a Ca2' transport system which is very labile. Although it may serve as a secondary "pump," it would be interesting to determine if and to what extent the "pump" and membrane as a whole are related to the onset of rigor mortis. Needless to say, these are speculative involve- ments of the sarcolemma in post-mortem muscle. However, it is clear an understanding of this membrane may lead to methods whereby the properties may be altered to increase the palatability and economic value of meat.

The functional importance of the muscle cell membrane is well recognized. Although isolation procedures have been available for a long while, we have just begun to scratch the surface of elucidating the properties of this membrane and their relationship to the overall function of the cell. Needless to say, there is still much to learn about this dynamic membrane in living and post-mortem muscle.

As eluded to throughout this paper, there are some obstacles which must be criticall evaluated when working with this membrane system. These include: (13 defining the system, (2) development of reliable markers for the sarcolemma per se and for the quantitative assessment of impurities from other membranous and non-membranous elements, and (3) increasing our understanding of present isolation procedures in order to minimize impurities in the final preparation. These are just some problems inherent not only to working with the sarcolemma but also with other membranous systems (see Warren and Glick, 1971). Future research will undoubtedly clarify these points and at the same time provide us with a better understanding of the properties of this dynamic and beautiful membrane system. 334

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D. E. Goll: How does muscular dystrophy affect the properties of the sarcolem?

Cedric Matsushima: Surprisingly, the transport ATPase activity is slightly higher than normal; also, recent work has shown higher ammnts of sialic acid in the membranes from the dystrophic animal.

D. E. Goll: Are there any problems in preparing sarcolemma from postmortem muscle, or is it the same as preparation immediately after death?

Cedric Matsushima: The only study that comes close to this is that of Dr . Hultin . H. 0. Hultin: If you isolate it from post-rigor muscle it's much eaSier.

B. B. Marsh: Thank you very much, Cedric, for an interesting paper. The first paper of the second half of our program is concerned with the sarcoplasmic reticulum and its possible role in postmortem muscle. It's to be given by my colleague frm Wisconsin, Marion Greaser, whose name has already appeared in earlier papers today. A lot of questions were ultimately shelved until Marion's paper, so I'm sure he will be referring back to some of these earlier comments.