J. Gergely

Biochemical Abnormalities of the Sarcoplasmic Reticulum in Muscular Dystrophy

Abstract Calcium-uptake and ATPase studies carried out on fragmented sarcoplasmic reticulum from muscle of patients with Duchenne dystrophy showed low initial and total calcium uptake, low ATPase activity and normal efficiency, without any relation to the stage of the disease. In myotonic dystrophy, on the other hand, high initial and normal total calcium uptake and normal or higher than normal efficiencies were found. The data suggest early involvement of both fiber types in Duchenne dystrophy and primary involvement of red fibers in myotonic dystrophy. Actomyosin abnormalities found in three cases of Duchenne dystrophy indicate involvement of the contractile apparatus in the later stages of the disease. The only clinical-biochemical correlation observed in this study was the relation of the ATPase activity of the fragmented sarcoplasmic reticulum to the onset of Duchenne dystrophy.

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).

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.

Electron spin resonance of myosin spin labeled at the St thiol groups during hydrolysis of adenosine triphosphate

Abstract On the addition of Mg2+ and ATP the electron spin-resonance spectrum of the spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodoacetamide, selectively bound to the S1 thiol groups of myosin changes from the one characteristic of strong immobilization to one indicating weaker immobilization. The latter spectrum persists during the steady state of hydrolysis of ATP; when hydrolysis is complete it changes to a spectrum identical with that produced by ADP. This third spectrum indicates a mobility between that of the label on myosin in the absence of ATP and that found during the steady state. The same results are obtained with heavy meromyosin or subfragment-1. The appearance of the spectrum typical of the steady state requires the presence of a divalent cation; either Ca2+ or Mg2+ is effective. It also seems to require hydrolysis of ATP since it is not observed in the absence of activating cations, when hydrolysis has been inhibited with N-ethylmaleimide, or when nonhydrolyzable analogs of ATP are used. One of these, β,γ-imino-adenosinetriphosphate, produces the same spectral change as ADP. These different spectra have been interpreted in terms of the kinetic scheme developed by Lymn and Taylor for native myosin in which the rate-limiting step follows the rapid hydrolysis of the terminal phosphate of ATP. Ourpresent observation of an “initial burst” of Piliberation with S1-labeled myosin justifies the application of this scheme. According to this scheme the intermediate responsible for the steady-state spectrum contains the products of ATP hydrolysis but its spectrum is distinct from the complex formed by adding products. This suggests the presence of two spectrally distinct myosin-product complexes. The changes in esr spectra probably reflect localized conformational changes in the head of the myosin molecule. Reducing the pH, temperature, or salt concentration substantially reduces the mobility of the spin labels during the hydrolysis of ATP, suggesting that the conformation of myosin during the steady state may depend on the temperature, pH, and concentration of salt. Alternatively, the spectral changes may be brought about by a change in the relative concentrations of two or more spectrally distinct steady-state intermediates. Changes in these parameters have little or no effect on spectra recorded in the absence of substrate.

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.

The binding of Cu2+ to actin without loss of polymerizability: The involvement of the rapidly reacting SH group

Abstract G-or F-actin binds 1 mole of Cu 2+ per mole of actin without denaturation and without replacement of the essential Ca 2+ or Mg 2+ . The association constant is ~10 16 . Denaturation of actin, e.g., by EDTA treatment, abolishes the high-affinity Cu 2+ binding. The absorption spectrum of Cu-actin shows a new intense absorption band centered at 347 nm (ϵ = 3000 M −1 cm −1 ) and a weak band centered at 550 nm (ϵ = 130 M −1 cm −1 ). The tryptophyl fluorescence of actin is strongly (70%) quenched by the binding of Cu 2+ . Circular dichroic spectra show multiple bands associated with each absorption band indicating an asymmetrical environment for the bound Cu 2+ and suggest that histidine is one of the ligands. Electron-spin resonance studies show that Cu 2+ maintains its valence when bound and also suggest that there are approximately three nitrogen ligands. Chemical modification studies of the sulfhydryl groups provide evidence for the involvement of one SH group in the binding of Cu 2+ to actin.

The stoichiometry of the reaction of the spin labeling of F-actin and the effect of orientation of spin-labeled F-actin filaments

Abstract The electron spin resonance (ESR) spectra of actin from rabbit skeletal muscle spin labeled with nitroxide analogs of N -ethylmaleimide were studied. When N -(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide was added to F-actin there was a preferential reaction of 1 mole of label per 46,000 g of actin. The ESR spectrum of oriented films of spin labeled F-actin varied depending on the orientation with respect to the applied magnetic field, suggesting that the label had a preferred orientation on the actin monomers. The 2pπ orbital on the N-atom that holds the unpaired electron of the spin label was more nearly parallel than perpendicular to the helical axis of F-actin. It was confirmed that spin labeling did not inhibit the G-F transformation and that during polymerization of G-actin spin labels became more strongly immobilized. On reaction of maleimide spin labels having varying chain lengths with F-actin both strongly and weakly immobilized labels were observed and the proportion of weakly immobilized labels increased as the chain length was increased.

Studies on the Na+- and K+-activated adenosine triphosphatase in human striated muscle☆

The ATPase activity of the 10,000–30,000 g fraction obtained from human striated muscle homogenates, deoxycholate-treated, has been studied. This fraction contains a Mg++-activated ATPase that is insensitive to ouabain and inhibited by low concentrations of azide, oligomycin, and quinidine. A Na+,K+-activated ATPase is also present which is completely inhibited by ouabain, and its activity appears to be enhanced by the presence of azide, oligomycin, and quinidine. Similar concentrations of these three agents cause similar degrees of inhibition of the Mg++-activated ATPase activities of the 10,000–30,000 g fraction and of a “mitochondrial fraction” (1000–8000 g) further treated with deoxycholate. The Mg++-stimulated ATPase activity of these mitochondria is inhibited by Na+ plus K+. These experiments plus studies of mixtures of aged 10,000–30,000 g fraction and mitochondria suggest that the fresh 10,000–30,000 g fraction contains two ATPases, one present in mitochondrial fragments and the Na+,K+-stimulated one. The Na+ inhibition of the former, unless eliminated by aging or by mitochondrial ATPase inhibitors, masks the activity of the Na+,K+-stimulated ATPase. n nStudies on the aged 10,000–30,000 g fraction demonstrate that the Na+-K+ stimulated ATPase has a pH optimum at 7.5–8.0, is inhibited by calcium, PCMB, and high concentrations of oligomycin and quinidine. Na+ could not be replaced by other monovalent cations, while K+ could be replaced by NH4+, Cs+, Rb+, and Li+, the order being that of decreasing effectiveness.

Changes in conformation and nucleotide binding of Ca, Mn, or Mg-G-actin upon removal of the bound divalent cation: Studies of ultraviolet difference spectra and optical rotation

Abstract The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca2+, Mn2+, or Mg2+ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn2+ > Ca2+ > Mg2+. ATP release is partially reversible upon Ca2+ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca2+, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca2+, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.

Effect of ions on sarcoplasmic reticulum fragments.

Abstract Fragments of cardiac sarcoplasmic reticulum (FSR) were prepared from homogenates of dog hearts and skeletal FSR from rabbit white skeletal muscle by differential centrifugation. The effect of substituting NO 3 − , I − , and SCN − for Cl on the ATPase and Ca uptake of fragmented dog cardiac sarcoplasmic reticulum (grana) was studied. Calcium uptake and ATPase were depressed in the order SCN − > I − > N0 3 − , the sequence found for skeletal FSR (2). The effects of the divalent cations Zn 2+ and UO 2 2+ were studied on both rabbit white skeletal muscle and dog cardiac FSR; 1 m m Zn 2+ and uranyl depressed calcium uptake of both preparations. The results are discussed in terms of the effects of these ions on cardiac and skeletal muscle contraction and current views of excitation-contraction coupling.