Removal Of Light Chains Research Articles

Involvement of 17K dalton light chain of smooth muscle myosin in substrate-induced conformational change.

Conformational changes of the region involving 17K dalton light chain (G2) in gizzard myosin induced by ATP and its analogs were studied by a combination of light chain exchange and fluorescence spectroscopy. The cysteinyl residues of myosin light chain mixture, 20K dalton G1 and 17K dalton G2 chain, were labeled with a fluorescent thiol reagent, N-(1-anilionon-aphthyl-4ymaleimide (ANM). Chicken gizzard myosin was incubated with a large excess of the fluorescence labeled exogenous light chains in 2 M urea. After removal of urea and unbound light chains, a strong fluorescent band was observed at the position of the 17K dalton light chain (G2) of myosin on SDS PAGE. There was no difference in enzymatic activity and light chain composition between myosins before and after light chain exchange. The emission spectrum of ANM label in 17K dalton light chain in the bound state in myosin showed a fluorescence enhancement and a blue shift on adding ATP but not ADP. The spectral change disappeared after conversion of ATP to ADP. Various nucleoside triphosphates other than ATP had similar effects on the fluorescence spectrum. The relative effectiveness of most nucleotides on the fluorescence spectral change of ANM in the light chain bound in myosin agreed well with that on tryptophan residues in myosin, except in the case of ADP and CTP. These results suggest that 17K dalton light chain of gizzard myosin is closely related to the ATPase site of myosin.

Myosin-Linked Calcium Regulation in Squid Mantle Muscle Light-Chain Components of Squid Myosin1

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.

Plasmapheresis in the treatment of acute renal failure in multiple myeloma

Three patients with multiple myeloma and severe acute renal failure were treated by repeated plasmapheresis. Recovery of renal function was observed in all. The pathogenetic role of light chains and the possible mechanisms responsible for renal damage are discussed. It is suggested that the removal of light chains by plasmapheresis may be of therapeutic value in this condition.

Changes in crossbridge attachment in a myosin-regulated muscle.

THE accompanying paper by Simmons and Szent-Gyorgyi1 presents a direct confirmation that regulation of activity in scallop muscles is mediated by the thick filaments: when one particular polypeptide (a regulatory light chain) is removed from the myosin molecules, tension development is no longer inhibited by the absence of calcium ions. Yet we still have no picture of the structural events associated with myosin-linked regulation, comparable with (for example) the ‘tropomyosin-shift’ model for the interactions of calcium, troponin and tropomyosin in actin-linked regulation (see, for example, Wakabayashi et al.2). We report here that removal of the regulatory light chain can lead to a structural change observed by X-ray diffraction. Our observations on the scallop may have a more general significance since myosin-linked regulation is found, in addition to actin-linked regulation, in many types of muscle3.

Reversible loss of calcium control of tension in scallop striated muscle associated with the removal of regulatory light chains.

THE control by calcium of the ATPase activities of myosin, actomyosin and myofibrils from molluscan muscles requires the presence of a particular ‘regulatory’ light chain of myosin1,2. In these muscles troponin is not found in stoichiometric amounts and does not function as a major regulatory component2,3. In scallop muscles one of the two regulatory light chains per myosin is readily and reversibly removed by reducing the divalent cation concentration with EDTA2,4, with the result that the preparations become ‘desensitised’ to calcium, that is, the ATPase activity is maximal whether or not calcium is present. ‘Resensitising’ can be achieved by the recombination of regulatory light chains with myosin or myofibrils. We have extended these studies to a scallop muscle preparation (chemically ‘skinned’ fibre bundles) in which the contractile machinery is intact, and we show here that the regulatory light chains can be removed and recombined, with a concomitant loss and recovery of calcium control over tension production.

Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.

Physico-chemical studies on the light chains of myosin III. Evidence for a regulatory role of a rabbit myosin light chain

Treatment of myosin with DTNB causes a decrease in the calcium sensitivity of actomyosin, concurrently with the release of the DTNB light chains. The removal of the calcium-binding DTNB light chains is accompanied by a loss of the calcium binding capacity of myosin. A regulatory role is ascribed to these light chains.

Stoichiometry and Sequential Removal of Light Chains of Myosin

The experiments described below are designed to answer the following questions: (a) What is the stoichiometry of the light chains per mole of myosin? and (b) Is it possible to remove sequentially one light chain at a time from the myosin molecule, and, if possible, to correlate the effect of such sequential removal with the ATPase activities of myosin?