Frank A. Sreter

Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin.

Abstract The two essential thiol groups of myosin, SH-1 and SH-2, have been localized in an ~ 20K segment of the heavy chain by analysis of the distribution of radioactivity after tryptic digestion of tryptic heavy meromyosin (HMM) or papain-HMM subfragment-1, both labeled at SH-1 and SH-2 with [14C]iodoacetamide and [14C]N-ethyl maleimide, respectively. The results are discussed in the framework of earlier work (Balint, M., Sreter, F. A., Wolf, I., Nagy, B., and Gergely, J. (1975) J. Biol. Chem. 250, 6168–6177) on the tryptic fragmentation of myosin heavy chain and in the light of more recent work on the location of a fragment that reacts with a photoaffinity analog of ATP (Szilagyi, L., Balint, M., Sreter, F. A., and Gergely, J. (1978) Fed. Proc. 37, 1695) and of suggestions concerning the binding of ATP in the region containing the SH-1 and SH-2 (Elzinga, M., and Collins, J. H. (1977) Proc. Nat. Acad. Sci. USA74, 4281–4284).

Structural and functional changes of myosin during development: Comparison with adult fast, slow and cardiac myosin

Abstract ATPase (Ca2+ and K+ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO4 polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC2 except that the relative amount of LC3 is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.

Temperature, pH and seasonal dependence of Ca-uptake and ATPase activity of white and red muscle microsomes

Abstract The total Ca ++ -uptake of fragmented sarcoplasmic reticulum (FSR) of either white or red skeletal muscle was optimal between pH 6.5–7.0 and 5.7–6.4 in the presence or absence of oxalate, respectively. Both the total Ca ++ -uptake and the initial rate of uptake were considerably lower in red FSR than in white FSR. The Ca ++ -uptake ability of either FSR showed seasonal variations; in the summer the activity was considerably less than during the winter or springtime. Under optimal conditions Ca ++ -uptake in the absence of oxalate was 250 nmoles/mg for white and 40 nmoles/mg for red FSR; corresponding uptakes in the presence of oxalate were 6.0 and 1.2μmoles/ mg. The initial rates measured over the first seconds were 1.8 and 0.15μmoles/mg/min for white and red FSR, respectively, regardless whether oxalate was present or absent. The initial rate of the “basal” ATPase activity of particulate or solubilized red FSR was almost one order of magnitude higher than that of white FSR. The latter, however, showed a significant calcium dependence while the red FSR ATPase was insensitive to calcium. The temperature dependence of Ca ++ -uptake was the same for the two types of FSRs; the activation energies were 11 and 25 kcal/mole in the absence and in the presence of oxalate, respectively. The activation energy of the hydrolysis of ATP in the absence of Ca ++ (basal ATPase) was the same for both FSR (11 kcal/mole); Ca ++ roughly doubled the activation energy for white, but had little or no effect on red FSR. 32 P incorporation from γ- 32 P-ATP was eight times less for red FSR than for white FSR.

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles.

The time-course of Ca2+ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Several methods were used to trigger Ca2+ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca2+ concentration ([Ca2+0]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca2+ release induced by either halothane or Ca2+ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca2+ released by halothane at low [Ca2+0] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca2+ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca2+ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum (k = 83 s-1) than in normal (k = 37 s-1). Thus, all types of Ca2+ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca2+ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.

Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit straited muscles

Abstract We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The β subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the α subunit of tropomyosin gradually increases so that in adult skeletal muscles the α subunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the α subunit which remains invariant with development. Two subspecies of the α subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC 3 light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles.

Tryptic digestion and localization of calcium uptake and ATPase activity in fragments of sarcoplasmic reticulum

Fractions sedimented at 25,000 g for 50 minutes from homogenates of mouse hind leg muscles were prepared according to the procedure of Von Korff originally designed to obtain intact cardiac mitochondria (41). Negatively stained preparations of this fraction (fragmented sarcoplasmic reticulum) appear to be essentially free of contamination with mitochondrial fragments and myofilaments. Vesicles consisting of a globular head with a diameter of 0.1–0.2 μ and one or more tail-like portions with a roughly constant width of 350 and a varying length of 0.05–1 μ (most frequently 0.2 μ) predominate. The surface of the vesicle, both of the head and tail portion, is covered with particles approximately 40 in diameter and spaced 50–80 from each other, the center being 40–60 from the surface of the vesicular membrane. Ca deposits, in the presence of oxalate, were localized in the globular portion. ATPase activity, as judged electron microscopically from the deposition of lead phosphate, was also localized in the globular portion, very frequently at the junction with the tail. Digestion of vesicles with trypsin caused changes in both the Ca uptake and ATPase activity. With a trypsin-vesicular protein ratio of 1:25 to 1:100 a rapid drop in Ca uptake was accompanied by an increase in ATPase activity. With even lower (1:300) ratios, a rapid initial drop in Ca uptake was eventually followed by a slower phase of decrease; the ATPase in this case increased monotonically with an inflection point subsequent to the rapid drop in Ca2+ uptale. The digestion caused structural disruption of the vesicle, first in the tail, and eventually led to swelling of the globular portion and the appearance of areas devoid of regularly arranged particles on the membrane.

Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin.

Photoaffinity labelling of tryptic and chymotryptic heavy meromyosin with 3′O-3-[N-(4-azido-2-nitrophenyl) amino]propionyl-adenosine 5′-triphosphate (arylazido-β-alanine ATP) resulted in incorporation of radioactivity and inhibition of the ATPase activity. ATP prevented the reaction with the photoaffinity label, as shown by the lack of incorporation of 3H and intact ATPase activity. On the tryptic digestion of either type of photoaffinity labeled HMM the label was found in a 25K peptide identifiable with the N-terminus of the myosin heavy chain (Lu et al., Fed. Proc. 37 1695 1978). The results are discussed in the light of previous localization of the reactive thiol groups, SH-1 and SH-2 (Balint et al., Arch. Biochem. Biophys. 190, 793 1978).

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. II. The effects of dantrolene on Ca2+ release induced by caffeine, Ca2+ and depolarization

The effects of dantrolene, which is a known muscle relaxant, on Ca2+ release from the isolated sarcoplasmic reticulum induced by several different methods [1) addition of caffeine, (2) Ca2+ jump, and (3) membrane-depolarization produced by choline chloride replacement of potassium gluconate) were investigated. Dantrolene inhibited caffeine-induced Ca2+ release with C1/2 = 2.5 microM, whereas there was no effect on Ca2+ release induced by a Ca2+ jump. The amount of Ca2+ released by depolarization was reduced if Ca2+ release was triggered in an earlier phase of the steady state of Ca2+ uptake (time elapsed between the addition of ATP and the triggering of Ca2+ release, tATP less than 4 min); while, if triggered in a latter phase (tATP greater than 4 min) dantrolene enhanced depolarization-induced Ca2+ release. C1/2 for the inhibition and that for enhancement of depolarization-induced Ca2+ release were 1.0 and 0.3 microM, respectively. These results suggest that dantrolene affects several different steps of the mechanism by which Ca2+ release is triggered. The sarcoplasmic reticulum and T-tubule membrane fractions had 7.9 nmol dantrolene-binding sites/mg (Kassoc = 1.0 X 10(5) M-1) and 21.0 nmol/mg (Kassoc = 1.1 X 10(5) M-1), respectively. The time-course of dantrolene binding to sarcoplasmic reticulum was monophasic, while that to T-tubules was biphasic.

Structural features of the surface of the vesicles of FSR—Lack of functional role in Ca2+ uptake and ATPase activity☆

Abstract Negative staining with uranyl acetate of the vesicles of fragmented sarcoplasmic reticulum from rabbit skeletal muscle has revealed three structural components in the vesicle wall: (1) the membrane portion proper, (2) spheres with a diameter of about 40 A on the exterior surface of the vesicle membrane, and (3) stalks with a width of about 20 A connecting the spheres with the membrane. Digestion with trypsin (at a 1:20 trypsin-vesicle protein ratio by weight) in a medium containing 0.1 m KCl (pH 7.0) with or without added sucrose leads to the removal of both surface spheres and stalks leaving smooth vesicles. If 4 n m calcium is included during digestion, only the surface spheres disappear and vesicles attached to the exterior surface by what appears to be a brush border result. The previously reported loss of Ca 2+ uptake and increase in ATPase activity of vesicles during tryptic digestion are prevented by adding high concentrations of sucrose (1.0 m ) but not the removal of spheres and stalks. The capacity of forming the phosphorylated intermediate by the transport enzyme from ATP remains intact in these vesicles. Similarly, vesicles devoid of only the surface spheres obtained by digestion in the joint presence of sucrose and calcium have retained essentially intact activities. The results indicate that all important activities related to the Ca 2+ transport mechanism are located in the membrane portion proper of the vesicle wall, and that the surface spheres and stalks have little or no role to play in Ca 2+ transport.

Fragmentation of myosin by papain—studies on myosin from adult fast and slow skeletal and cardiac, and embryonic muscle

Abstract Digestion of insoluble myosin with soluble papain produces heavy meromyosin subfragment 1 (HMM-S-1) having ATPase activity and the ability to combine with actin. These fragments of myosin do not undergo appreciable changes in ATPase activity, chromatographic behavior, or actin combining ability during digestion up to 2 h but, as shown by sodium dodecyl sulfate gel electrophoresis, several splits occur in both the heavy and light polypeptide chains. The largest fragment of heavy chain present in fast, slow, cardiac and embryonic HMM-S-1 has a mass of 89,000 daltons. This fragment undergoes further degradation resulting in fragments having masses of the order of 70,000, 50,000, and 27,000 daltons. The latter fragment and other material resulting from the proteolysis of myosin appear as bands in that region of the gels where the light chains are found in electrophoretograms of the parent myosin. The precise size of the fragments and the rates of their formation depend on the type of myosin; slow and cardiac HMM-S-1 and their fragments show greater stability. Embryonic myosin has properties intermediate between those of fast skeletal and cardiac myosin. Experiments involving the combination of HMM-S-1 with actin and experiments with glutaraldehyde cross linking and chromatography on Sephadex G-200 indicate that the fragments separated by sodium dodecyl sulfate gel electrophoresis are held together by noncovalent forces in HMM-S-1.