An improved assay for nanomole amounts of inorganic phosphate.

A colorimetric assay for the determination of nanomole amounts of inorganic phosphate is described. The procedure combines a very high molar extinction with color stability and insensitivity to newly released phosphate from labile organophosphates.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.     

Mechanism of stimulation by epinephrine of active transepithelial Cl transport in isolated frog cornea

Summary In the isolated frog cornea, the effects of 0.1mm epinephrine were measured on both the transepithelial and intracellular electrical parameters. Epinephrine increased the short-circuit current (I _sc) and transepithelial electrical conductance (g _t) by 176 and 96%, respectively. The effective electromotive driving force for active transepithelial Cl transport (E _Cl) was 45 mV and agrees with the value forE _Cl calculated by a different technique in the isolated rabbit corneal epithelium (Klyce, S.D., Wong, R.K.S., 1977,J. Physiol. (London) 266: 777). With respect to the tear-side bathing solution, epinephrine caused the intracellular potential difference of shortcircuited frog corneas to decrease from –54 to –50 mV (P>0.05). The fractional resistance of the apical membrane {F(R _o)=(R_o/R_o+R_i)} whereR _o andR _i represent the resistances of the apical and basolateral membranes, respectively, decreased from 0.38±0.06 to 0.23±0.03. Using these values ofF(R _o) and the cellular conductances, the calculated Cl resistances ofR _o andR _i decreased 4.3- and 2.3-fold, respectively. However, the value forE _Cl calculated from the intracellular electrical measurements (48 mV) did not appear to change since this value was in close agreement with the value forE _Cl calculated from the effects of epinephrine on the transepithelial electrical parameters. Thus, the effects of epinephrine onI _sc andg _t can be accounted for by increases in the Cl conductance of both the apical and basolateral membranes. Epinephrine caused the potential difference across the basolateral membrane to hyperpolarize by 9 mV. All of these results are consistent with the notion that the steps in transepithelial Cl transport include uphill movement into the cell across the basolateral membrane followed by downhill movement across the apical membrane into the tear-side bathing solution.     

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.     

Implications of an anomalous intracellular electrical response in bullfrog corneal epithelium

Summary The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential differenceV _sc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl₂ (K⁺ channel blocker). With Ba²⁺ the depolarization of theV _sc by 25mm K⁺ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceR _o remained unchanged, the relative basolateral membrane resistanceR _i, which includes the lateral intercellular space, increased at the most by less than twofold after Ba²⁺. These effects in conjunction with the depolarization of theV _sc by 62 mV after increasing the stromal-side K⁺ from 2.5 to 100mm in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a=R _o/R_i) from 13 to 32 are all indicative of an appreciable basolateral membrane K⁺ conductance. This ratio decreased significantly after exposure to either 25mm K⁺ or Ba²⁺. The decline ofR _o/R_i with 25mm K⁺ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K⁺, but rather perhaps a larger decrease ofR _o thanR _iAlso an increase of lateral space resistance may offset the effect of decreasingR _i with 25mm K⁺. In contrast,R _o/R_i did transiently increase during voltage clamping of the apical membrane potential differenceV _o and exposure to 25mm K⁺ on the stromal side. This increase and subsequent decrease ofR _o/R_i supports the idea that increases in stromal K⁺ concentration may produce secondary membrane resistance changes. These effects onR _o/R_i show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na⁺ withn-methyl-glucamine in Cl-free Ringer's on the stromal side hyperpolarized theV _sc by 6 mV whereas 10^–4 m ouabain depolarized theV _sc by 7 mV. Thus the basolateral membrane contains K⁺, Na⁺ and perhaps Cl^– pathways in parallel with the Na/K pump component.     

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.     

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.     

Hybrid myosin light chains containing a calcium-specific site from troponin C.

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction [Reinach, F. C., Nagai, K. & Kendrick-Jones, J. (1986) Nature 322, 80-83]. To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes'. In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.     

Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle

Isoforms of C-protein in adult chickens which differ in fast (pectoralis major, PM) and slow (anterior latissimus dorsi, ALD) skeletal muscles can be distinguished immunochemically with monoclonal antibodies (McAbs) specific for the respective fast (MF-1) and slow (ALD-66) protein variants (Reinach et al., 1982 and 1983). The expression of these C-proteins during chick muscle development in vivo has been analyzed by immunoblot and immunofluorescence procedures. Neither MF-1 nor ALD-66 reacted with whole-cell lysates or myofibrils from PM of 12-day-old embryos. However, both McAbs bound to peptides of 145 kDa in PM from late embryonic and young posthatched chickens. All of the myofibers in these muscles reacted with both antibodies, but the binding of the anti-slow McAb (ALD-66) diminished progressively with age and was completely negative with PM by 2 weeks after hatching. In contrast, the ALD muscle from 17 days in ovo thru adulthood only reacted with ALD-66; no binding of MF-1 could be detected at these stages. Since both fast and slow myosin light chains (LC) coexist within embryonic pectoralis and ALD muscles (e.g., G. F. Gauthier, S. Lowey, P. A. Benfield, and A. W. Hobbs, 1982, J. Cell Biol.92, 471–484) yet segregate to specific fast and slow muscle fibers at different stages of development, the temporal transitions of C-protein and myosin LC were compared during myogenesis. “Slow-type” C-protein appeared after the disappearance of slow myosin light chains, whereas the accumulation of the “fast-type” light chains occurred before the expression of “fast-type” C-protein. The pattern of isoform transitions appears to be far more complex than previously suspected.     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.