A proposed mechanism for myosin magnesium adenosine triphosphatase activity

A formal mechanism for the myosin MgATPase is proposed. The basic characteristics of this mechanism require that the binding of substrate at either one of two equivalent nucleotide sites of uncomplexed myosin prevents binding of substrate at the other unoccupied site (i.e. negative cooperativity) and that the rapid formation of a myosin-product complex permits binding of substrate at the unoccupied site. Analogue computer kinetic simulations indicate that the proposed mechanism is compatible with the observed transient phase kinetics characterizing the interaction of the enzyme with MgATP. In addition, analysis of the derived rate equation show that the mechanism is also consistent with existing steady-state kinetic data for the myosin MgATPase. A simpler mechanism is proposed for the subfragment-1 MgATPase that is shown to be compatible with the existing kinetic data. Features of the proposed myosin MgATPase mechanism are incorporated into a model of contraction which utilizes the bipartite structure and nucleotide site interaction of the myosin crossbridge to provide an efficient utilization of ATP in the contraction cycle.     

A steady-state kinetic study of the ribonuclease A catalyzed hydrolysis of uridine-2′:3′(cyclic)-5′-diphosphate

Initial velocity measurements were made on the ribonuclease A catalyzed hydrolysis of P-5′-Urd-2′:3′-P in the pH range 4.0–8.0 at 25 °C in 0.1 m Tris-acetate/0.1 m KCl. The pH dependence of the Michaelis constant, K_m, the turnover number k_s, and $\text{k}{}_{\mathrm{s}}\text{K}{}_{\mathrm{m}}$ for P-5′-Urd-2′:3′-P were similar to those reported for Urd-2′:3′-P (5). When P-5′-Urd-2,3-P and Urd-2′:3′-P were compared under similar conditions the average difference in k_s and K_m indicated that these parameters were 5-fold and 23-fold lower, respectively, for P-5′-Urd-2′:3′-P. The slight difference in the pH dependence of $\text{k}{}_{\mathrm{s}}\text{K}{}_{\mathrm{m}}$ for these two substrates can be interpreted in terms of a specific interaction of the enzyme at the 5′ position of P-5′-Urd-2′:3′-P, which permits a less exclusive dependence on the ionized state of the free enzyme in binding this substrate. The nature of the interaction of the substrate 5′-phosphomonoester group with the enzyme is discussed in terms of possible interactions with Lys-41 and His-119.     

Spermine stabilization of folded ribonuclease T1

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.     

Rapid kinetic studies on the conformation of single-stranded DNA

The conformational states of single-stranded calf thymus DNA were probed using the tmeperature-jump kinetic technique. A rapid decrease in transmission at 260 nm followed by a broad relaxation spectrum was observed for single-stranded DNA at 0.15M KCI, pH, 6.5 at 25°. The relaxation spectrum could also be detected as a hydrogen ion release. Sonicated samples of 103,000 daltons showed identical relaxation kinetics as unsonicated samples. The relaxation kinetics were studied as a function of temperature, pH, and salt concentration and could be interpreted as representing the melting of short, isolated folded segments. The rapid process that could not be kinetically resolved is interpreted as single-strand unstacking. Upon the addition of excess MgCl₂ at pH 7.0, a hydrogen ion release from single-stranded DNA was observed that was 3.5 times greater than that observed for a comparable sample of native DNA. Evidence is presented which indicates that charge-pairs exist in folded segments that are A-T rich. There was no kinetic evidence of structures in single-stranded DNA other than single-stranded stacking and short isolated folded regions.