D.J. Hartshorne

Composition of the myosin light chain kinase from chicken gizzard.

Abstract The Ca 2+ -dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca 2+ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events.

The effect of phosphorylation of gizzard myosin on actin activation.

Abstract Gizzard myosin is phosphorylated by a kinase found in chicken gizzards. The 20,000 dalton light chains are the only subunits to show an appreciable extent of 32P incorporation. Phosphorylation requires trace amounts of Ca2+. The Mg2+-ATPase activity of gizzard myosin in the phosphorylated form is activated to an appreciable extent by skeletal actin, whereas the activation of the non-phosphorylated myosin is verylow. These results suggest that the Ca2+-sensitive regulatory mechanism of gizzard actomyosin is mediated via a kinase. In the presence of Ca2+ the onset of contraction and the resultant increase of the Mg2+-ATPase activity we suggest is due, at least partly, to the phosphorylation of the 20,000 dalton light chains. Whether or not Ca2+ binding by myosin is also essential remains to be established.

A relationship between Ca2+ sensitivity and phosphorylation of gizzard actomyosin.

Summary Ca 2+ regulated actomyosin from chicken gizzard is phosphorylated to a significant extent (∼2.5 moles P per 10 6 g. actomyosin) only in the presence of Ca 2+ . The Ca 2+ dependence of phosphorylation is similar to that shown by the ATPase activity. When the actomyosin is desensitized to the effects of Ca 2+ the phosphorylating system is removed. Reconstitution of Ca 2+ sensitivity is accompanied by an increase in the extent of phosphorylation. These results suggest a relationship between phosphorylation and the Ca 2+ sensitive ATPase activity of gizzard actomyosin.

Fractionation of troponin into two distinct proteins

Abstract The protein complex which governs the calcium sensitivity of natural actomyosin (Ebashi, 1963) has been shown to consist of two components, tropomyosin and troponin ( Ebashi and Kodama, 1966 ; Hartshorne and Mueller, 1967 ). In the present study troponin has been separated into two distinct proteins, as judged by amino acid analyses. The fractionation was achieved at low pH and high ionic strength. One of the fractions, termed troponin B, effected a calcium insensitive inhibition of synthetic actomyosin ATPase activity, which was enhanced by the addition of tropomyosin. The other fraction, troponin A, conferred calcium sensitivity to the troponin B - tropomyosin system. The separation was reversible and all the properties of the source troponin were regained upon mixing troponin A and B in the correct proportions.

Calcium binding by the troponin complex, and the purification and properties of troponin A.

Abstract The percentage of tropomyosin and troponin in rabbit psoas muscle was estimated to be 4.2% and 5.6% of the myofibrillar protein, respectively. This suggests that on the thin filament there are two molecules of tropomyosin per 390 A, and probably two molecules of the troponin complex. Approximately 60% of the calcium bound by troponin was accounted for by the component which conferred the calcium sensitivity, troponin A. The binding data for troponin A indicated two classes of binding sites. The Scatchard plots were approximated by assuming that each class contained 4.8 sites/10 5 g protein and that the high affinity binding constant was equal to 5.4 · 10 6 M −1 and the low affinity constant was equal to 2 · 10 5 M −1 . Troponin A was purified by column chromatography and it was found that both the calcium binding ability and activity in the assays with troponin B were confined to one component. This was characterized by its high mobility on disc electrophoresis. The purified troponin A was roughly spherical, and had a molecular weight of approximately 18 500. It is unusual in that it contained a high proportion of phenylalanine relative to tyrosine. The excess of negatively charged amino acids would also suggest that at neutral pH it was a polyanion. On a weight basis the purified troponin A bound approximately 50% more calcium than the unpurified troponin A.

Studies on troponin

Abstract Troponin was isolated from preparations of the ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA)-sensitizing factor over the pH range of 3.5–4.6. The variations among troponins were due essentially to differences in the proportion and properties of the troponin A component. This was dictated by the choice of the source fraction (EGTA-sensitizing factor) and by the efficiency of recovery of troponin A in troponin by the isoelectric fractionation. A higher yield of troponin A was obtained at the alkaline edge of the given pH range. The relative proportion of troponin A in troponin was reflected by the A278 mμ:A260 mμ ratio and by the effect troponin had on the Mg2+-activated ATPase activity of synthetic actomyosin. The inhibitory effect of the EGTA-sensitizing factor on the Mg2+-activated ATPase activity of synthetic actomyosin in the presence of EGTA, was due to an interaction of troponin B and tropomyosin. Ultracentrifugal analysis revealed that this interaction was reflected by the formation of a hypersharp boundary different from the boundaries of troponin B or tropomyosin alone. Inhibition of the ATPase activity and complex formation was maximal at a ratio of tropomyosin to troponin B of approx. 1:1 (w/w). It is proposed that troponin A is responsible for the calcium sensitivity of the EGTA-sensitizing factor.

The effect of temperature on actomyosin.

Abstract 1. The effect of temperature on the ATPase activity of actomyosin systems was studied. The Arrhenius plot for natural actomyosin was curved between o °C and 40 °C for the Mg 2+ -activated ATPase activity in the presence of calcium. A similar plot in the absence of calcium was linear over the same temperature range. The Arrhenius plot of the Mg 2+ -activated activity of desensitized actomyosin in both the presence and absence of calcium was linear up to 40 °C. The addition of troponin B and tropomyosin to desensitized actomyosin did not alter the linearity of the plots. However, when troponin A was added (to complete the regulatory system) a non-linear plot resulted. It is proposed that the characteristic curvature of the Arrhenius plots for the Mg 2+ -activated ATPase activity of natural actomyosin and myofibrils is a consequence of the binding of calcium to troponin A. 2. At temperatures above 40 °C all of the systems studied demonstrated a nonlinearity, which was not influenced to any great extent by the regulatory proteins. 3. As the temperature was increased above 20 °C calcium sensitivity became less and at 50 °C was lost. It is unlikely that this effect was due to either the oxidation of critical sulfhydryl groups or to an alteration of the calcium binding characteristics of troponin. A possible explanation is suggested.

Sulfhydryl groups of natural actomyosin essential for the Ca2−-sensitive response: location and properties

Abstract 1. 1. Certain sulfhydryl groups in natural actomyosin are essential for Ca2+ sensitivity. These are located on the heavy chains of the myosin molecule. 2. 2. Reaction of these sulfhydryl groups is influenced by ATP and ATP analogs. ATP exhibits a pronounced protective effect, with ADP and pyrophosphate being less effective. 3. 3. The critical sulfhydryl groups are most susceptible to reaction at low ionic strength and in the presence of actin. 4. 4. The reaction of this site does not produce the same effects as the reaction of the S1 sulfhydryl groups. 5. 5. The inhibition of ATPase activity by troponin B or troponin B plus tropomyosin is also removed under conditions similar to those which eliminate Ca2+ sensitivity.

Actin crosslinked with glutaraldehyde: Evidence to suggest an active role for actin in the regulatory mechanism

Abstract Actin crosslinked with glutaraldehyde retains the ability to activate the Mg 2+ -ATPase activity of heavy meromyosin subfragment 1, but the resultant ATPase activity is not controlled by the regulatory proteins, troponin and tropomyosin. Fluorescent energy transfer measurements imply that the crosslinked actin is frozen in the active state. These results indicate that the conformation of actin is important in the regulatory mechanism, and suggest that actin plays a more active role in this mechanism than thought previously.

ATPase activity and superprecipitation of skeletal muscle actomyosin of frog and rabbit: effect of temperature on calcium sensitivity.

Abstract 1. 1. In the presence of Mg 2+ , but not Ca 2+ , the activation energy of rabbit skeletal actomyosin ATPase was constant from 0 to 40°C. With the addition of a trace amount of Ca 2+ , the activation energy was reduced by almost a factor of three at temperatures greater than 20°C. 2. 2. For frog skeletal actomyosin ATPase the activation energy decreased by a factor of two at temperatures greater than 15°C. This reduction in activation energy occurred both in the presence and absence of Ca 2+ and, therefore, did not require the binding of Ca 2+ to troponin, as was the case for rabbit actomyosin. 3. 3. At temperatures greater than 15°C there was a progressive reduction in the Ca 2+ requirement for actomyosin superprecipitation. In the presence of 1 mM MgATP as substrate the Ca 2+ sensitivity of frog actomyosin was eliminated at 30–35°C while that of rabbit actomyosin was eliminated at 40–45°C. 4. 4. A simple model to explain the effect of temperature on Ca 2+ sensitivity is proposed and the implication of these findings for the mechanical behavior of muscle is considered.