Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites

Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.     

Electrostatics and electrodynamics of bacteriorhodopsin

The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent dipole directed perpendicular to the plane and an induced dipole in the plane. Fits of the orientation function to data measured at low field strengths demonstrate: an increase of the permanent dipole moment mu with the square of the disk radius r2, whereas the polarizability alpha increases with r4; the ionic strength dependence is small for mu and clearly stronger for alpha; the permanent dipole moment is 4x10(6) D at r = 0.5 micron. According to the risetime constants, the induced dipole does not saturate and increases to 4x10(8) D at 40 kV/cm and r = 0.5 micron. The data indicate that the permanent dipole is not of some interfacial character but is due to a real assymetry of the charge distribution. The experimental dipole moment per protein monomer is approximately 55 D, whereas calculations based on the structure of Grigorieff et al. (Grigorieff, N., T.A. Ceska, K.H. Downing, J.M. Baldwin, and R. Henderson. 1996. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259:393-421) provide a dipole moment of approximately 570 D. The difference is probably due to a nonsymmetric distribution of charged lipid residues. It is concluded that experimental dipole moments reflect the mu-potential at the plane of shear for rotational diffusion, in analogy to the sigma-potential used for translational diffusion. It is suggested that the permanent dipole of bacteriorhodopsin supports proton transport by attraction of protons inside and repulsion of protons outside of the cell. Dichroism rise curves at field strengths between E = 150 and 800 V/cm reveal an exponential component with time constants tau 3r in the range between 1 and 40 ms, which is not found in Brownian dynamics simulations on a disk structure using hydrodynamic and electric parameters characteristic of bacteriorhodopsin disks. The experimental data suggest that this process reflects a cooperative change of the bacteriorhodopsin structure, which is induced already at a remarkably low field strength of approximately 150 V/cm.     

Chemomechanical transduction in the actomyosin molecular motor by 2',3'-dideoxydidehydro-ATP and characterization of its interaction with myosin subfragment 1 in the presence and absence of actin

The effect of torsional freedom about the N-glycoside bond of ATP in the ability of the nucleoside triphosphate to support chemomechanical transduction (Takenaka et al., 1978) has been investigated by examining the ability of the nucleotide analogue 2',3'-dideoxy-2',3'-didehydro-ATP (1b, enf-ATP) to act as a substrate for myosin subfragment 1 in the presence and absence of actin and to support actin sliding in the standard in vitro motility assay. By converting the ribosyl ring of the natural substrate to the rigid and almost planar enofuranosyl ring, effects on torsional freedom about the N-glycoside bond due to changes in ribosyl ring pucker and/or by steric interferences of the protons attached to the 2' and 3' carbons are eliminated allowing for increased torsional freedom about the N-glycoside bond. The data indicate that this enofuranosyl analogue is an excellent substrate for subfragment 1 and actosubfragment 1 and produces actin sliding velocities which are twice as fast as those observed with ATP in the standard in vitro motility assay. The analogue diphosphate is trapped in S1 by the common P(i) analogues, but the rate of formation of the ternary complex formed with Vi is very slow compared to that observed with MgADP. Similar conformations of S1 are formed with Mg.enf-ATP and MgATP under steady-state conditions, but S1 with bound Mg.enf-ADP differs significantly from that observed with MgADP.     

The density of a homogeneous population of cells controls resetting of the program for swarmer formation in the unicellular marine microorganism Noctiluca scintillans

Radiofrequency transitions within K = 2 asymmetry doublets have been observed for the CO2-CO van der Waals complex. A Stark effect measurement on the J = 2, K = 2 transition provides an electric dipole moment of μ = 0.2493(1) D. Combining this result with the permanent moment of CO, μCO = 0.1098 D, gives a change of moment on complex formation of Deltaμ = 0.140 D. The sign of Deltaμ is such that the CO end of the complex is more positive than CO2. The origin of Deltaμ should not be attributed to any single mechanism, and several different contributions to Deltaμ are discussed. Copyright 1998 Academic Press.     

Characterization of rash with indinavir in a national patient cohort

FTIR absorption spectra of the fundamental bands of LiF and LiCl were measured with an apodized resolution of 0.01 cm-1. The Herman-Wallis analysis of these spectra led to the determination of the ratio of the dipole derivative to the permanent dipole moment, [μe/(dμ/dr)ere], for each molecule. The vibrational dependence of the dipole moment for each molecule has been already reported and these data were reanalyzed to determine the dipole moment function independently. The results from the Herman-Wallis analysis were compared with these results, and good agreement was found. This demonstrates that the Herman-Wallis analysis can produce reliable relative values of dipole moment functions. Copyright 1999 Academic Press.     

Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state

The crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE), both with a transition state analog (MgADP x AIF4-) in the active site, have been determined to 2.9 A and 3.5 A resolution, respectively. The MDE structure with an ATP analog (MgADP x BeFx) was also determined to 3.6 A resolution. In all three structures, a domain of the C-terminal region, the "converter," is rotated approximately 70 degrees from that in nucleotide-free skeletal subfragment 1 (S1). We have found that the MDE-BeFx and MDE-AIF4- structures are almost identical, consistent with the fact that they both bind weakly to actin. A comparison of the lever arm positions in MDE-AIF4- and in nucleotide-free skeletal S1 shows that a potential displacement of approximately 10 nm can be achieved during the power stroke.     

Calorimetric studies of the thermal unfolding of smooth muscle myosin fragments and their complexes with ADP and phosphate analogs

The thermal unfolding of turkey gizzard smooth muscle myosin subfragment 1 (S1) and heavy meromyosin (HMM) in the absence of added nucleotides, in the presence of ADP, and in S1 or HMM ternary complexes with ADP and Pi analogs, orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-), have been studied by differential scanning calorimetry (DSC). It has been shown that the formation of these ternary complexes causes significant structural changes in S1 or in the heads of HMM which are reflected in a pronounced increase of the protein thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. Phosphorylation of regulatory light chains (RLC) in S1 or in HMM had practically no influence on these effects. In general, the changes caused by various Pi analogs in smooth muscle S1 or HMM were similar to those observed earlier with skeletal muscle S1 devoid of RLC. It is concluded that RLC and their phosphorylation do not significantly affect the character of structural changes induced in motor domains of the HMM heads by the formation of ternary complexes HMM--ADP--Vi, HMM--ADP--AlF4-, and HMM--ADP--BeFx--stable analogs of the intermediate states of the HMM ATPase reaction, HMM.ADP.Pi and HMM. ATP.     

The effects of focal and diffuse brain damage on strategy application: evidence from focal lesions, traumatic brain injury and normal aging

The use of symmetric reversing electric field pulses in electrooptic studies of rigid macromolecules in order to determine the ratio between the permanent and the induced dipole moments is well established. Application of this method to studies of small macromolecules requires a field reversal time of only a few nanoseconds. No high current pulse generator capable of producing symmetric kV pulses with such a short reversal time is available for studies of small macromolecules in physiological salt solutions, but it has long been known how to make such reversing pulses that are asymmetric. In order to take advantage of the opportunity offered by the latter fact, we here present a theoretical analysis in the thermal domain of the electrooptic properties of solutions containing rigid macromolecules with axial symmetry when exposed to asymmetric reversing electric field pulses. The analytical expressions needed for quantitative determination of the ratio between the permanent and the induced electric dipole moments of rigid macromolecules using electrooptic data obtained employing reversing electric pulses with given asymmetry are presented. The feasibility of this new approach is demonstrated by including experimental electric birefringence data for a 12 kDa protein (segment 14 of alpha-spectrin from Drosophila brains) in near physiological salt solutions obtained using a coaxial cable pulser producing 2 microseconds long pulses with a reversal time of about 15 ns.     

Latex allergy

The purpose of this study was to assess the mechanical properties--torsional moment, maximum angular deflection, maximum bending moment, and permanent angular deflection--of four brands of nickel-titanium (NiTi) endodontic file, and compare them with a conventional stainless-steel instrument, both in the presence and absence of sodium hypochlorite (NaOCl). NiTi instruments from four manufacturers were randomly selected and subjected to NaOCl treatment for 12 or 48 h, or not at all. The mechanical properties under test were then measured automatically by a digital torque memocouple. Torsional moment and maximum angular deflection indicate the resistance to torsional fracture of an instrument, maximum bending moment the stiffness of the instrument, and permanent angular deflection the strength of the base alloy. All instruments evaluated complied with or exceeded ADA/ANSI Specification No. 28, with the sole exception of the Maillefer ISO size 40 for torsional moment. JS Dental and McSpadden NiTi files were the most resistant to torsional fracture, but all NiTi files were inferior when compared with stainless-steel files from a previous study. However, NiTi files were superior in flexibility, and Maillefer and Brasseler instruments were the best of the instruments tested. NiTi files also had negligible permanent deformation angles. Furthermore, for all properties tested, NaOCl had no statistically significant effect.     

Interaction of rabbit C-reactive protein with phospholipid monolayers studied by microfluorescence film balance with an externally applied electric field

C-reactive protein (CRP) is one of the most characteristic acute-phase proteins in humans and many other animals. It binds to phosphorylcholine in a calcium-dependent manner. In addition, CRP activates the complement systems via the classical pathway. The interaction between rabbit CRP (rCRP) and model biological membrane is studied using dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine monolayers. Observations with fluorescence microscopy indicate that rCRP is more likely to be incorporated in the liquid phase of monolayers. Such incorporation does not depend on the presence of calcium and is not inhibited by phosphocholine. The area occupied by the protein when incorporated into the monolayer was estimated. The dipole moment density of the protein crossing the air/water interface was measured by applying an external electric field. Our results indicate that calcium binding leads to a conformational change in CPR, which might modify the orientation of CRP in the monolayer. In addition, a negative charge or negative difference in dipole moment density facilitates the incorporation of CPR into the monolayer.     

The preference of tryptophan for membrane interfaces

One of the ubiquitous features of membrane proteins is the preference of tryptophan and tyrosine residues for membrane surfaces that presumably arises from enhanced stability due to distinct interfacial interactions. The physical basis for this preference is widely believed to arise from amphipathic interactions related to imino group hydrogen bonding and/or dipole interactions. We have examined these and other possibilities for tryptophan's interfacial preference by using 1H magic angle spinning (MAS) chemical shift measurements, two-dimensional (2D) nuclear Overhauser effect spectroscopy (2D-NOESY) 1H MAS NMR, and solid state 2H NMR to study the interactions of four tryptophan analogues with phosphatidylcholine membranes. We find that the analogues reside in the vicinity of the glycerol group where they all cause similar modest changes in acyl chain organization and that hydrocarbon penetration was not increased by reduction of hydrogen bonding or electric dipole interaction ability. These observations rule out simple amphipathic or dipolar interactions as the physical basis for the interfacial preference. More likely, the preference is dominated by tryptophan's flat rigid shape that limits access to the hydrocarbon core and its pi electronic structure and associated quadrupolar moment (aromaticity) that favor residing in the electrostatically complex interface environment.     

X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPgammaS, and MgADP are reported at 2.1, 1.9, and 2.1 A resolution, respectively. Crystals were obtained by cocrystallization and were isomorphous with respect to those of S1dC. MgADP.BeFx [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972]. In all three structures, the electron density for the entire nucleotide was clearly discernible. The overall structures of all three complexes are very similar to that of the beryllium fluoride complex which suggests that the differences in the physiological effects of ATPgammaS and AMPPNP are due to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower affinity of AMPPNP for myosin. In S1dC.MgAMPPNP, the presence of the bridging nitrogen prompts the side chain of Asn233 to rotate which disrupts the hydrogen bonding pattern in the nucleotide binding pocket and alters the water structure surrounding the ribose hydroxyl groups. It appears that this change is responsible for the reduced affinity of AMPPNP for myosin relative to ATPgammaS. In contrast to the G-proteins, there is no major change in the conformation of the ligands that coordinate the nucleotide in S1dC.MgADP. This is due to three water molecules that adopt the approximate positions of the three oxygens on the gamma-phosphate and maintain the interactions with the Mg2+ ion and protein molecule. Interestingly, the thiophosphate group is evident in S1dC.MgATPgammaS even though it is slowly hydrolyzed by myosin. This suggests that the conformation observed here and in chicken skeletal myosin subfragment-1 [Rayment, I., et al. (1993) Science 261, 50-58] is unable to hydrolyze ATP and represents the structure of the prehydrolysis weak binding state of myosin.     

Adhesion molecule expression and endothelial cell activation in cutaneous leukocytoclastic vasculitis. An immunohistologic and clinical study in 42 patients

We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram-negative bacterium Serratia marcescens using finite-difference Poisson-Boltzmann methods for electrostatic calculations and a bead-model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double-stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (-1 e) charge per monomer at pH 7. The most dramatic shift in pKa involves His 89 whose pKa increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments.     

Species-specific primers resolve members of Fusarium section Fusarium. Taxonomic status of the edible "Quorn" fungus reevaluated

This paper concerns rotational energy levels and line intensities for electronic, vibrational, and microwave transitions in an open-shell complex consisting of an open-shell diatomic molecule and a closed-shell partner. The electronic state of the open-shell diatomic fragment is a 2S+1Sigma state, where S >/= 12, the close-shell partner could be a rare gas atom or a diatomic molecule or a planar polyatomic molecule. We are considering a near-rigid rotor model for a nonlinear complex, taking into account thoroughly all effects of the electron spin and the quartic centrifugal distortion correction terms. The total Hamiltonian is expressed as H=Hrot+Hsr+Hss+Hcd+Hsrcd+Hsscd. We have derived all the nonvanishing matrix elements of the Hamiltonian operators in the molecular basis set. The rotational energy levels are calculated by numerical diagonalization of the total Hamiltonian matrix for each J value. The nonvanishing matrix elements of the electric dipole moment operator are derived in the molecular basis set for electronic, vibrational, and microwave transitions within the complex. Expectation values of the quantum numbers and of the parities of the rotational states are derived in the molecular basis set. Relative intensities of the allowed rotational transitions, expectation values of the quantum numbers and the parities are calculated numerically in the space of the eigenvectors obtained from diagonalization of the Hamiltonian matrix. The formalism and the computer program of this paper are considered as extensions to our previous work [W. M. Fawzy and J. T. Hougen, J. Mol. Spectrosc. 137, 154-165 (1989); W. M. Fawzy, J. Mol. Spectrosc. 160, 84-96 (1993)] and are expected to be particularly useful for analyzing and fitting high-resolution spectra of weakly bonded oxygen complexes. A brief discussion of the Hamiltonian operators, the matrix elements, and the computer program is given. Copyright 1998 Academic Press.     

Diphtheria and poliomyelitis incidence in Europe

Measurements are presented of angular velocities of rotation of mammalian cells of K562 (human) and SP2 (mouse) in external alternating electric fields over a frequency range of 0.5 kHz to 12 MHz. Electro-rotation of the cells was observed for the case of "two cells in contact' using two parallel, cylindrical electrodes; only one cell was located on the electrode. A theoretical analysis is also presented which shows that the cell rotation arises from a torque produced by the interaction between the primary electric dipole moment induced in the spinning cell and the secondary electric fields, generated by the primary dipole induced in the adjacent cell. These secondary fields are out of phase with the applied electric field. The results show that (a) only the cell not located on the electrode rotates, (b) maximal electro-rotation occurs at two different excitation field frequency domains for the frequency range employed here, (c) the spin speed of the rotating cell at each frequency domain is much less than the excitation frequency, (d) the rotation direction of the cell depends on the angle (theta) between the external electric field and the line joining the centres of the two cells and (e) for a given angle theta, the rotation direction is the same for both excitation frequency domains. The experimental measurements allowed us to estimate the conductivities of the cytoplasms and membrane capacitances of the cells of K562 and SP2. The conductivities of the cytoplasms of the cells of K562 and SP2 were estimated to be 0.2 and 0.3 Sm-(1), respectively, whereas the membrane capacitances of these cells were found to be 2.7 +/- 0.8 and 9.8 +/- 0.6 mFm-(2), respectively.     

Cooperative binding of ATP and MgADP in the sulfonylurea receptor is modulated by glibenclamide

The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.     

Analysis of Effect of Ultrasound on the Magnetic Topography of Electroplated Ni Films by Magnetic Force Microscopy (MFM)

It has been shown to be extremely beneficial to employ power ultrasound to assist electrochemical processes. Considerable mass transport has been demonstrated with the application of ultrasound. The magnetic microstructure, magnetic phase, and orientation images show better magnetic particle deposits in the presence of a sonication environment. Furthermore, deposits with ultrasound show permanent dipole moment magnetization. Hence, Ni film synthesis in presence of ultrasound may open new heights for hard disk drives.     

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator

Chloroplast mutator (chm) of Arabidopsis is a recessive nuclear mutation that causes green and white variegation in leaves and is inherited in a non-Mendelian fashion. In this study, we have identified and characterized a mutant observed in F1 and backcrossed BC1 populations from a cross between chm1-3 and ecotype Columbia. This mutant, maternal distorted leaf (MDL), grows very poorly and is distinguished by distorted rough leaves and aborted flowering organs. Electron microscopic observation showed that in MDL plants, a significant portion of mitochondria are abnormal and appear to be nonfunctional. DNA gel blot and sequence analysis of the MDL mitochondrial DNA (mtDNA) revealed rearrangements in two mtDNA fragments associated with rps3-rpl16 genes (encoding ribosomal proteins S3 and L16, respectively). One rearrangement resulted in the insertion of the rps3-rpl16 operon downstream of atp9. An independent deletion in this region had eliminated the majority of rps3. In contrast, another rearrangement deleted part of rpl16, whereas rps3 remained intact. RNA gel blot analysis indicated that expression of these genes is also altered as a consequence of the mtDNA rearrangements. Thus, a mutation at the CHM locus affects mitochondrial gene expression, and impaired mitochondrial function may result in the distorted phenotype.     

MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle

Tonic rabbit femoral artery and phasic rabbit ileum smooth muscles permeabilized with Triton X-100 were activated either by increasing [Ca2+] from pCa > 8.0 to pCa 6.0 (calcium-ascending protocol) or contracted at pCa 6.0 before lowering [Ca2+] (calcium-descending protocol). The effects of, respectively, high [MgATP]/low [MgADP] [10 mM MgATP + creatine phosphate (CP) + creatine kinase (CK)] or low [MgATP]/[MgADP] (2 mM MgATP, 0 CP, 0 CK) on the "force-[Ca]" relationships were determined. In femoral artery at low, but not at high, [MgATP]/[MgADP] the force and the ratio of stiffness/force at pCa 7.2 were significantly higher under the calcium-descending than calcium-ascending protocols (54% vs. 3% of Po, the force at pCa 6.0) (force hysteresis); the levels of regulatory myosin light chain (MLC20) phosphorylation (9 +/- 2% vs. 10 +/- 2%) and the velocities of unloaded shortening V0 (0.02 +/- 0.004 l/s with both protocols) were not significantly different. No significant force hysteresis was detected in rabbit ileum under either of these experimental conditions. [MgADP], measured in extracts of permeabilized femoral artery strips by two methods, was 130-140 microM during maintained force under the calcium-descending protocol. Exogenous CP (10 mM) applied during the descending protocol reduced endogenous [MgADP] to 46 +/- 10 microM and abolished force hysteresis: residual force at low [Ca2+] was 17 +/- 5% of maximal force. We conclude that the proportion of force-generating nonphosphorylated (AMdp) relative to phosphorylated cross-bridges is higher on the Ca2+-descending than on the Ca2+-ascending force curve in tonic smooth muscle, that this population of positively strained dephosphorylated cross-bridges has a high affinity for MgADP, and that the dephosphorylated AMdp . MgADP state makes a significant contribution to force maintenance at low levels of MLC20 phosphorylation.     

Crystallographic structure of an intact IgG1 monoclonal antibody

The structure of an intact monoclonal antibody for phenobarbital, subclass IgG1, has been determined to 3.2 A resolution by X-ray crystallography. The molecule was visualized in a monoclinic unit cell having an entire immunoglobulin as the asymmetric unit. The two Fab segments, both with elbow angles of 155 degrees , were related by a rotation of 179.7 degrees plus a translation along the approximate dyad of 9 A. This is the first observation of such an Fab translation in a structurally defined antibody. The approximate 2-fold of the Fc was independent of that relating Fabs, making an angle of 107 degrees with the Fab dyad. The angle between long axes of the Fabs was 115 degrees, the most acute angle yet observed, yielding a distorted Y shaped molecule. This is in contrast to the distorted T shape of the only other intact IgG (2a) whose complete structure is known. Primary lattice interactions arise through formation of VH antiparallel beta ribbons whose strands are contributed by pseudo dyad related H2, and by L3 hypervariable loops from neighboring molecules. While one CH2 domain was mobile, Fabs and three domains of the Fc were well defined, as were hinge polypeptides connecting Fabs to the Fc, and the covalently attached oligosaccharides. Direct interactions are observed between hinge polypeptides, the glycosylated loop of one CH2 domain, and the oligosaccharide. Lattice interactions clearly influence, perhaps even determine the overall conformation of the antibody observed in this crystal. Comparison of this IgG1 with previously determined intact antibody structures extends the conformational range arising from segmental flexibility.