Eur. J. Biochem. 93, 113-121 (1979)

Properties of Microsomal Subfractions Isolated from Developing Rabbit Skeletal Muscle Elibieta ZUBRZYCKA, Marek MICHALAK, Danuta KOSK-KOSICKA, and M. Gabriela SARZALA Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Experimental Biology, Warsaw (Received March 13/October 10, 1978)

A heterogeneous muscle microsomal fraction isolated from 3 - 4-day-old rabbits was separated into two subfractions by continuous sucrose-density-gradient centrifugation. The two subfractions differ from each other morphologically and biochemically. Negative staining reveals that the heavier subfraction (U2) consists of 'thin-contour' vesicles resembling those of sarcoplasmic reticulum membrane from mature muscle. The surface of many of them (about 60 %) is covered by 4-nm particles. The lighter subfraction ( U l ) contains smooth vesicles with a contour thicker than that of the vesicles of subfraction U2. This difference is probably due to the high lipid content in the vesicles of subfraction U1. The extraction of loosely bound lipids (60 % of total lipid content) with anhydrous diethyl ether changes the ultrastructure of these vesicles; their contour becomes thin and the vesicles resemble those in subfraction U2. The activity of Mg2'-dependent ATPase is very high in subfraction U1, but this subfraction lacks calcium uptake and Ca2'-dependent ATPase activity. Contrary to that, subfraction U2 shows the activity of Ca2'-dependent ATPase and calcium-loading capacity in the presence of oxalate. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the protein pattern of both subfractions is more complicated than that of sarcoplasmic reticulum vesicles from adult muscle. Both subfractions contain proteins of molecular weight between 12000 and 105000. The antibody precipitation technique indicates the presence of Ca" -dependent ATPase in subfraction U2 and the presence of calsequestrin in both inicrosomal subfractions.

Sarcoplasmic reticulum, an intracellular membrane system in muscle, has a relatively simple structure and chemical composition [l]. Its function in the regulation of the calcium concentration in the sarcoplasm during the contraction-relaxation cycle is well defined [2,3]. These features make it a unique model for studies on the biogenesis and assembly of biological membranes. In our previous studies [4,5] we have shown using sodium dodecyl sulphate/polyacrylamide gel electrophoresis that microsomes isolated from undeveloped rabbit skeletal muscle contain mainly proteins of molecular weight between 30000 and 80000. The Ahbreviation. EGTA, ethyleneglycol-bis-(2-aminoethyl ether)-

N,N,N',N '-tetraacetic acid. Enzymes. ATPase or adenosinetriphosphatasc (EC 3.6.1.3); glucose-6-phosphatase or u-glucose-6-phosphate phosphorylase (EC 3.1.3.9); acetylcholinesterase or acetylcholine hydrolase (EC 3.1.I .7); 5'-nucleotidase or 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5); glycogen phosphorylase (EC 2.4.1.1); cytochrome c oxidase or ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.1).

105000-M, protein, corresponding to the Ca2 -dependent ATPase, the major protein in sarcoplasmic reticulum from mature muscle, is only one of the minor bands in undeveloped muscle. On the other hand, calsequestrin and high-affinity calcium-binding protein, two extrinsic sarcoplasmic reticulum proteins of molecular weight 45 000 and 55 000, respectively, have been found in microsomal vesicles at very early developmental stages [4,5]. From these results we have concluded that both extrinsic proteins are probably synthesized earlier than the ATPase. A population of microsomal vesicles, obtained from undeveloped muscle by the method used for isolation of sarcoplasmic reticulum vesicles from mature muscle, was however morphologically heterogeneous. This could imply that the microsomal fraction contained vesicles originating not only from newly synthesized sarcoplasmic reticulum membrane, but also from other membranous systems. For this reason in -I

114

Microsomal Subfractions from Developing Muscle

Table 1. Distribution of specific activities of marker enzymey and glycogen content in microsamal fractions from undeveloped muscle Values reported are averages with the number of determinations shown in parentheses Fraction

5'-Nucleotidase

Na+,K+ATPase

Glucose-6phosphatase

nmol pi x mg protein-' x min-' ~

Acetylcholinesterase

Phosphorylase b

Glycogen

nmol acetylcholine split x m g protein-' x min-'

pmol Pix mg protein x min-'

mg/mg protein

~

'

Unfractionated microsomal fraction

51.6 (3)

15.0 ( 3 )

3.6 (3)

5.0 (3)

1.5 (6)

0.025 (6)

u1

61.3 (5)

35.0 ( 3 )

4.5 (5)

7.0 (3)

4.5 (7)

0.150 (7)

u2

15.0 (6)

0.0 (3)

0.0 (6)

0.5 (3)

0.0 (7)

0.000 (7)

our previous paper [4,5] we have called this fraction the 'microsomal fraction'. Recently we have been able to show [6,7] that the fraction of sarcoplasmic reticulum vesicles from mature muscle can be separaxed into two subfractions by an additional sucrose-density-gradient centrifugation. In this paper a very similar fractionation procedure has been used to separate the heterogeneous microsoma1 fraction from undeveloped muscle into subfractions. Each subfraction has been characterized morphologically and biochemically with the special attention paid to the identification of the ATPase and calsequestrin. MATERIALS AND METHODS Preparation of Muscle Microsomal Fractions

The heterogeneous microsomal fraction sedimenting between 8000 - 50000 x g was isolated from skeletal muscle of 3 - 4-day-old rabbits according to the procedure described previously [4]. This heterogeneous fraction was suspended in 20 mM imidazole buffer, pH 7.2 and 100 mM KCI (called throughout this paper imidazole-KC1 buffer) at a protein concentration of 8 mg per ml, and 5 ml of the suspension was applied to the top of a 30 ml continuous sucrose-density gradient (8.8 - 64.4