Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle.

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

The new anticancer drug [(2R)-aminomethylpyrrolidine](1, 1-cyclobutanedicarboxylato)platinum(II) and its toxic S enantiomer do interact differently with nucleic acids.

The interaction of the two chiral isomers of the new anticancer agent [Pt(ampyr)(cbdca)] (ampyr=aminomethylpyrrolidine, cbdca=cyclobutanedicarboxylate) with 5'-GMP and with short G-containing oligonucleotides has been studied using (1)H and (31)P NMR, UV-vis spectroscopy and molecular modelling. Each isomer loses the cbdca ligand upon binding to the DNA fragments. Two geometrical isomers of the DNA adducts are formed owing to the presence of the unsymmetric ampyr ligand. These isomers prove to be GG-N7,N7 chelates for d(GpG), d(pGpG) and d(CpGpG). A slight preference for the formation of one geometrical isomer is found in the case of DNA fragments having a phosphate moiety and/or a C base at the 5'-site of the GG sequence. H-bonding interactions from the NH(2) moiety towards the 5'-phosphate group and/or the O atom of the C base clearly favour the formation of one geometrical isomer. The presence of these H-bonds, together with the bulky pyrrolidine ring, has resulted in the unique observation (by (1)H NMR) of NH protons of coordinated amines that do not rapidly exchange in a 99.95% D(2)O solution. Temperature-dependence studies show an extremely slow stack <--> destack conformational change for the CGG adducts of the S isomer, which could be related to these stable H-bonds of the amine protons towards the oligonucleotide. For the R isomer this stack <--> destack conformational change is faster, probably owing to more steric hindrance of the pyrrolidine ring as deduced from the NOESY data, and as also suggested by molecular modelling. The observation of extremely slow rotation around the Pt-N7 bond for [Pt(R-ampyr)(GMP-N7)(2)] provides further evidence for increased steric hindrance of the R isomer compared to the S isomer. The rate of binding of the drug to G bases proved to be second order for both isomers; in fact the (toxic) S isomer is about two times more reactive than the (non-toxic) R isomer, as seen from k(2) values of 0.17+/-0.01 M(-1)s(-1)for [Pt(S-ampyr)(cbdca)] and 0.09+/-0.01 M(-1)s(-1) for [Pt(R-ampyr)(cbdca)]. No solvent-assisted pathway is involved in these reactions, since the complexes prove to be stable in solution for weeks and therefore only a direct attack of the G base on the Pt must be involved. Because hardly any intermediate species can be detected during the reaction, coordination of the second G base must occur much faster than the binding of the first G base. Since direct attack of the nucleobases takes place, steric interactions become extremely important and therefore are likely to determine the reactivity, activity and even the toxicity of such Pt complexes.

Phosphorylation of ribosomal protein L18 is required for its folding and binding to 5S rRNA.

Ribosomal protein L18 from Bacillus stearothermophilus (bL18) includes a previously unreported phosphoserine residue. The folded conformation of the protein is stabilized by the dianionic form of the phosphate group of that residue. In the absence of Mg2+, the pK(a) of the phosphate group is so high that the protein is not fully folded at pH 7. In the presence of Mg2+, its pK(a) drops significantly, and consequently the native conformation of bL18 becomes stable at pH 7 and the protein is able to bind to 5S rRNA. Dephosphorylated bL18 does not bind to 5S rRNA at neutral pH.

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG).

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.