Wilhelm Hasselbach

Interaction of actin with phalloidin:: Polymerization and stabilization of F-actin

The cyclic peptide phalloidin, one of the toxic components of Amanita phalloides prevented the drop of viscosity of F-actin solutions after the addition of 0.6 M KI and inhibited the ATP splitting of F-actin during sonic vibration. The data concerning ATP splitting are consistent with the assumption (a) that only 1 out of every 3 actin units of the filaments needs to be combined with phalloidin in order to suppress the contribution of these 3 actins to the ATPase activity of the filament and (b) that all actin units of the filaments can combine with phalloidin with a very high affinity. -halloidin did not only stabilize the actin-actin bonds in the F-actin structure but it also increased the rate of polymerization of G-actin to F-actin. The ability of F-actin to activate myosin ATPase was not affected by phalloidin. The tropomyosin-troponin complex did not prevent the stabilizing effect of phalloidin on the F-actin structure.

ATP synthesis by the reverse of the sarcoplasmic calcium pump

The result of the release studies described in the preceeding report [l] prompted the search for net ATP synthesis by the sarcoplasmic calcium pump. It is shown that under the conditions where a fast ADP and inorganic phosphate dependent calcium release takes place from calcium loaded sarcoplasmic vesicles, the net outward movement of calcium is stoichiometrically related to a net formation of ATP. The experimental conditions are given in the legend to fig. 1.

Activation of calcium efflux by ADP and inorganic phosphate

The release of calcium from sarcoplasmic vesicles preloaded with calcium oxalate or calcium phosphate has been studied under various conditions such as the addition of high concentration of EGTA to ATP containing calcium uptake media or the depletion of EGTA containing uptake media from ATP [l-3] Under both conditions, the observed initial release rates are unidirectional flux rates because the calcium activity in the solution surrounding the vesicles is very low. When the vesicles were loaded with 300-500 nmoles calcium oxalate/mg of vesicular protein, the rates of release were found to be about 100 times slower than the initial rate of calcium uptake [ 1 ] . It has been shown that the very slow calcium efflux must be attributed to a low calcium permeability of the membranes, since the hydrolysis of membranal lipids or the binding of surface active agents induces a very fast calcium release. The effect of these membrane modifying treatments cannot be reversed. In this report it is shown that in contrast to these unspecific and irreversible permeability changes the calcium efflux is most specifically and reversibly activated by inorganic phosphate in combination with ADP. The experimental conditions are given in the legends to the figures.

Der physiologische Erschlaffungsfaktor und die Muskelgrana

1. 1. Practically the entire granule fraction which inhibits ATP-splitting and contraction is precipitated from crude rabbit-muscle extract by centrifuging at 25,000 × g for 1 h. The diameter of the granules in this fraction is about 1000 A (700–3000 A). 2. 2. The activity of the granules is irreversibly destroyed by SH-reagents, thymol and u.v. light. Inhibition caused by certain detergents, e.g. antarox is partially reversible. 3. 3. The activity of the granules is irreversibly lost when the granules are digested with several times their weight of trypsin in the absence of ATP, but is not affected when ATP is present. 4. 4. Before they can produce inhibition, the granules must be activated by ATP and Mg in the absence of Ca. Activation takes only a few minutes, and occurs normally during the experiments themselves. 5. 5. The inhibition of ATP splitting and contraction of fibrils or actomyosin floccules produced by the granules ceases as soon as the granules are removed. 6. 6. Thus, the granules do not withdraw any functionally essential substances from the contractile proteins, nor do they release any stable contraction-inhibiting substance to the actomyosin or to the solution. 7. 7. Over a wide concentration range, the effect of the granules does not depend on their absolute concentration, but on the granule/fibril ratio, in spite of the fact that the granules are not bound to the fibrils. Hence, the granules can not be identical with the inhibitor which reacts directly with the actin- and myosin-filaments. 8. 8. It seems probable, however, that the granules release a labile inhibitor, since their relaxing effect can be exhausted by repeated washing with fibril suspensions. During such washings, the fibrils remove no granules, but merely take away their activity. 9. 9. It is shown that all relaxing and inhibiting effects of the granules in actomyosin gels are brought about only by dissociation of the actomyosin in the presence of ATP and granules. The L-myosin-ATPase as well as the ability for and the state of polymerisation of the actin are not affected by the relaxing granules.

Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.

The inhibition of the sarcoplasmic calcium pump by prenylamine, reserpine, chlorpromazine and imipramine

Zusammenfassung1. Reserpin, Prenylamin, Chlorpromazin und Imipramin hemmen sowohl die Geschwindigkeit der Calcium-Aufnahme als auch die der Calcium-induzierten Extra-ATP-Spaltung an isolierten Vesikelmembranen des sarkoplasmatischen Reticulums von quergestreifter Muskulatur des Kaninchens. Die für eine 50%ige Hemmung erforderlichen Konzentrationen betragen beim Reserpin, Prenylamin und Chlorpromazin 3×10−5M, beim Impiramin ∼3×10−4M.2. Calcium-Efflux und -Influx werden durch die Pharmaka in gleichem Ausmaß gehemmt.3. Das Konzentrationsvermögen der Vesikel für Calcium wird deshalb durch die Hemmstoffe nicht beeinflußt.4. Der erste Reaktionsschritt des aktiven Calciumtransports der Vesikel — die Übertragung des Phosphates des ATP auf das Vesikelprotein — wird durch die Pharmaka nicht beeinflußt.Summary1. The calcium induced increase in ATPase and the rate of calcium uptake of the vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle are reduced by reserpine, prenylamine, chlorpromazine and imipramine. 3×10−5M reserpine, prenylamine, chlorpromazine and ∼3×10−4M imipramine are required to produce 50% inhibition of both activities.2. In the presence of the drugs the rate of calcium exchange at the cessation of calcium uptake is reduced to the same extent as the initial rate of calcium uptake.3. Neither the calcium storing capacity nor the ability to concentrate calcium are impaired by the drugs.4. The drugs do not affect the calcium dependent phosphate exchange between ATP and ADP and the calcium dependent formation of phosphoprotein.

Structural and chemical asymmetry of the calcium-transporting membranes of the sarcotubular system as revealed by electron microscopy

The calcium-transporting membrane of the sarcotubular system appears as a triple-layered asymmetric structure with a total thickness of about 60 A after fixation with osmium tetroxide or with glutaraldehyde followed by osmium tetroxide. In the vesicles that are formed upon isolation of the membrane, the peripheral opaque membrane component is denser and thicker than the opaque component facing the interior of the vesicles. This pattern is also observed in the membranes of intact muscle. In experiments where the vesicles were incubated with the SH-reagent Hg-phenyl azoferritin, a close packing of ferritin particles at the outer surface of the vesicular membrane was observed. After treatment with NEM, which blocks the SH-groups, no ferritin was attached to the membranes and the vesicles were clustered together. When the SH-groups at the active sites which are specifically involved in the calcium transport were selectively protected with ATP before NEM treatment, the ferritin was packed at the outer membrane surface as in normal material. The results have been interpreted as indicating a preferred localization of the active sites involved in the calcium transport at the outer surface of the membrane. The distance between adjacent sites seems to correspond exactly to the resolution of the method using ferritin as SHlabeling reagent. In a second series of experiments vesicular preparations were sonicated or allowed to age as well as treated with distilled water or alkali solutions in order to expose the inner membrane surface to the ferritin compound. Also in these preparations the label was located almost exclusively at the outer surface of the membrane, indicating that fewer reactive sites are located at the inner than at the outer surface.

Structural and enzymatic properties of the calcium transporting membranes of the sarcoplasmic reticulum

The mechanical and metabolic activity of all contractile tissue appears to be regulated by the intracellular calcium level. The key position of calcium in the regulation of muscular activity has been revealed mainly by the following observations: M, neither the contractile proteins in the living fibers, as well as in the extracted fibers, react enzymatically with ATP nor do they contract. (Weber & Herz, 1963; Portzehl et al., 1964). The contractile proteins are in the relaxed state. Actin and myosin filaments are dissociated. 2. Contraction and Actomyosin ATPase activity are induced when calcium is added to the isolated proteins or injected into the living fiber. Threshold contractions occur in both cases when the calcium level reaches 5 X M (FIGURE 1) (Heilbrunn & Wiercinsky, 1947; Portzehl et a]., 1964). 3. The amount of calcium required to saturate the isolated contractile proteins for maximal activity corresponds to one calcium ion per myosin molecule (Weber et al., 1963). The minimal amount of calcium which must be injected into the living fiber to produce maximal contraction is higher and more difficult to determine because the calcium injected is quickly removed from the contractile protein by the calcium pump of the sarcoplasmic reticulum (Heilbrunn & Wiercinsky, 1947; Caldwell & Walster 1963; Ashley et al., 1965). These findings are the main basis for the widely accepted hypothesis that activation of the contractile protein is caused by the liberation of calcium ions during excitation, and inactivation is brought about by calcium removal. In fibers with a short contraction-relaxation cycle (twitch fibers), the mechanical events follow the propagated action potential of the surface membrane within a few msecs. Therefore, one must assume that in these fibers calcium is released and removed inside of the contractile structure itself (Hill, 1948). The release of calcium is presumably triggered by the depolarization of the surface membrane. The depolarization may propagate to the interior of the fiber along transverse tubules, which are continuous with the outer membrane (Huxley, 1964; Franzini-Armstrong, 1964; Huxley, 1959). The tubules of the transverse system are flanked by the terminal cysternae of the sarcoplasmic reticulum. A high calcium concentration is maintained in the reticulum by a calcium pump localized in its membranes. It is tempting to assume that calcium is released from the cysternae in response to the depolarization of the transverse tubular system. A release from the transverse tubular system during depolarization is less likely but cannot be excluded. The latter mechanism would require a high concentration of calcium 1. If the free calcium concentration is below

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca2+ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca2+, Mg2+, MgATP2−, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml−1 SR was loaded actively with 0.02 mM45CaCl2, resulting in >90 nmol·mg protein−1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca2+, (2) the absolute amount of Ca2+ released by caffeine increases with the Ca2+ load of the SR, (3) increasing the ionic strength (μ) from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca2+ release increases with increasing (a) external Ca2+ concentrations up to 5 μM total Ca2+ (or 3 μM free Ca2+) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca2+ release is not affected by changes of either the Mg2+ or the MgATP2− concentration.

Die wechselwirkung verschiedener nukleosidtriphosphate mit aktomyosin im gelzustand

Abstract 1. 1. Splitting, tension and relaxing effect of nucleosidetriphosphate (NTP) are activated by magnesium ions. 2. 2. For each NTP the Mg ++ requirement is the same for splitting and tension. The Mg ++ requirement for the relaxing effect is much less. 3. 3. The amount of Mg ++ required for hydrolysis and tension increases from ATP to GTP in the order With more complete Mg ++ activation the development of tension decreases in the same order This also applies for the splitting (with the exception of acetyl-ATP). 4. 4. The extent of tension development always depends on the extent of the rate of splitting. It is thus immaterial whether or not the rates of splitiing are of varying magnitude since the splitting of the same NTP by Mg ++ is activated to a varying extent. Only acetyl-ATP develops with equal splitting less tension than the other NTPs. 5. 5. The Mg ++ requirement for the relaxing effect increases in the order: When the Mg ++ activation is maximal the relaxing effect decreases in the same order. 6. 6. The diversity of the NTP succession for tension and splitting on the one hand and for the relaxing effect on the other shows that considerable tension and splitting is produced when the NTP concerned contains an NH 2 group in the 6-positio — whether in the purine or the pyrimidine ring. The relaxing effect is by comparison greater with purine derivatives than with pyrimidine derivatives, independent of the presence of the NH 2 group in the 6-position in the ring system. 7. 7. The relaxing effect and tension development, in contrast to splitting, are not activated by Ca ++ ions. 8. 8. The “relaxing factor” ( Marsh ) works only in the presence of ATP in concentrations > 2 · 10 −3 M or of acetyl-ATP and CTP in concentrations −3 M . 9. 9. The “relaxing factor” does not, however, work in the presence of ITP, UTP and GTP in concentrations up to 10 −2 M . 10. 10. It is shown that the inter-action between actomyosin and NTPs does not depend on the fact that ATP is built up from the bound ATP of the actomyosin and the other NTP by means of nucleodiphosphokinase.