Structure of the 265-kilodalton complex formed upon EDC cross-linking of subfragment 1 to F-actin.

The conventional model of force generation in muscle requires the presence of at least two different contact areas between the myosin head (S1) and the actin filament. It has been found that S1 has two sites available for carbodiimide cross-linking, but it is generally believed that the myosin head can be cross-linked to only one actin through either site. We provide here, for the first time, evidence that one S1 can be cross-linked to two separate actin molecules. The covalent complex of one S1 with two actins was found to have an apparent molecular mass of 265 kDa. The formation of the 265-kDa acto-S1 complex was strongly dependent on the ratio of S1 to actin. Limited tryptic digestion converted the 265-kDa product into the 240-kDa complex by releasing a 27-kDa N-terminal S1 fragment. Limited subtilisin digestion of the 265-kDa covalent acto-S1 complex yielded 29-, 93-, and 66-kDa peptides which corresponded to the 29-kDa N-terminal domain of S1, actin-44-kDa (central domain of S1) and actin-22-kDa (C-terminal domain of S1) complexes, respectively. These peptides could be generated only if a single S1 has been cross-linked to two separate actins. The 265-kDa acto-S1 complex (S1:actin ratio = 0.5) had 60% of the ATPase activity of the 175-185-kDa acto-S1 complex (S1:actin ratio = 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Two different rigor complexes of myosin subfragment 1 and actin.

Our previous titration and cross-linking experiments showed that myosin subfragment 1 (S1) can bind to one or two monomers in F-actin [Andreev, O. A., & Borejdo, J. (1991) Biochem. Biophys. Res. Commun. 177, 350-356; (1992a) J. Muscle Res. Cell Motil. 13, 523-533; (1992b) Biochem. Biophys. Res. Commun. 188, 94-101]. In the present work we used a sedimentation method to extend these studies to equilibrium binding and a stopped flow method to investigate its kinetics. Both equilibrium and kinetic data indicated the existence of two different rigor complexes. On the basis of these data we developed a model which suggested that binding of S1 to F-actin occurred in two steps: (i) initial rapid binding to one monomer of F-actin, A + M<==>A.M and (ii) a consequent slow binding to a neighboring monomer, A.M + A<==>A.M.A, where A stands for actin and M for myosin subfragment 1. The second reaction can proceed only if the neighboring actin site is unoccupied. The model fit the equilibrium and kinetic binding data with equilibrium constants K1 = 6 x 10(6) M-1 and K2 = 4 and kinetic constants k+1 = 10.5 x 10(6) M-1 s-1, k-1 = 1.75 s-1, k+2 = 0.8 s-1, and k-2 = 0.2 s-1, where the subscripts refer to the reactions i and ii. These results corroborate our hypothesis that myosin head can make two types of complexes with F-actin and support our speculation that during a power stroke in contracting muscle a myosin head may first bind to one and then to two actins.

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.

[Study of the nature of determinants for synthesis of delta-endotoxin and beta-exotoxin in Bacillus thuringiensis subsp. thuringiensis strains--bitoxybacillin producers]. / Izuchenie prirody determinant sinteza delta-éndotoksina i beta- ékzotoksina v shtammakh Bacillus thuringiensis subsp. thuringiensis--produtsentakh bitoksibatsillina.

The growth of Bacillus thuringiensis subsp. thuringiensis strains (BT) 202, 1140, 98 producing bitoxybacillin in the presence of novobiocin, mitomycin C and at high temperature 43 degrees C resulted in obtaining of the mutants for the synthesis of crystalline protein (Cry) and exotoxin (Exo). Analysis of the plasmid content of the mutants has shown the Cry Exo phenotyre to correlate with the loss of 55 MD plasmid in the strain BT202 and the loss of 60MD plasmid in BT1140. The transfer of these plasmids into Bacillus cereus leads to the transfer of endo- and exotoxin production properties. The synthesis of Cry in BT98 is controlled by the 56MD plasmid, while the synthesis of Exo is encoded by the chromosome or plasmid that cannot be eliminated or transferred into other strains. Localization of Cry on the 55 and 56 MD plasmids in BT202 and BT98 is confirmed by the hybridization of the plasmid DNA with the DNA-probe.

[Plasmids for biodegradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine, and pyridine in strains of Arthrobacter]. / Plazmidy biodegradatsii 2,6-dimetilpiridina, 2,4-dimetilpiridina i piridina u shtammov Artrobacter.

Arthrobacter crysallopoietes strain KM-4 degrading 2,6-dimethylpyridine and strain KM-4a degrading both 2,6-dimethylpyridine and pyridine, Arthrobacter sp. KM-4b degrading 2,4-dimethylpyridine were isolated from soil. Arthrobacter crystallopoietes KM-4 and Arthrobacter sp. KM-4b contain 100 Md plasmids pBS320 and pBS323. Arthrobacter crystallopietes KM-4a harbours a 100 Md and 80 Md plasmids. Plasmid curing and conjugation transfer results confirm that these plasmids are involved in degradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine and pyridine. A mutant with lost ability to degrade 2,6-dimethylpyridine was isolated during the growth of strain KM-4 rifR at 42 degrees C. Electrophoretic analysis of the plasmid from temperature sensitive mutant revealed the deletion the size of 26 Md from pBS320 plasmid.

[Plasmids for biphenyl, chlorobiphenyl and metatoluylate degradation from Pseudomonas putida]. / Plazmida degradatsii bifenila, khlorbifenilov, meta-toluilata Pseudomonas putida.

Pseudomonas putida strain SU83, harbors the pBS311 plasmid coding for the degradation of biphenyl, 2- and 4-chlorbiphenyl, meta- and paratoluylate. The insertional mutants of the plasmid obtained by the transposon Tn5 insertion were isolated. One of the mutants was used for cloning of the biphenyl degradation genes. The plasmid pBS311:: Tn5 DNA was inserted into the BamHI site of the plasmid pBR322 and cloned. 11 recombinants of 354 tested were treated with 0.1% solution of 2,3-dioxybiphenyl. One of them has acquired the yellow colour testifying to conversion of 2,3-dioxyphenyl to "2-hydroxy-6-keto-6-phenylhexa-2,4-diene acid. The recombinant plasmid pBS312 from this clone is 10.5 kb in size, the size of the insert being 6.2 kb. Escherichia coli SU185 cells harbouring pBS312 are able to support metacleavage of 2,3-dioxybiphenyl, 3-methylcatechol and catechol, but not of 4-methylcatechol. The results suggest the cloned fragment to contain a gene for 2,3-dioxybiphenyl-1,2-dioxygenase, the third enzyme for biphenyl catabolism.

[Hybrid plasmid pBS251 containing genes for n-alkane degradation]. / Gibridnaia plazmida pBS251, soderzhashchaia geny degradatsii n-alkanov.

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.

[Oxidation of n-alkanes by Pseudomonas aeruginosa strain carrying the plasmid pBS251]. / Okislenie n-alkanov shtammom Pseudomonas aeruginosa, nesushchim plazmidu pBS251.

Pseudomonas aeruginosa PAO8 cannot use n-alkanes or their respective alcohols as a sole carbon source. However, it can grow on n-alkanes when plasmid pBS251 is transferred into its cells. The hybrid plasmid pBS251 is a plasmid RP4 containing genes which control the capability to grow on n-alkanes of the C6-C12 series. Studies of n-alkane oxidation by P. aeruginosa PAO8 carrying pBS251 have shown that this plasmid controls the inducible alkane and alcohol oxidizing activities; the subsequent steps of n-alkane oxidation controlled by chromosomal genes are constitutive.

[Resistance to organic tin compounds mediated by plasmids of bacteria of Pseudomonas genus]. / Determiniruemaia plazmidami bakterii roda Pseudomonas ustoichivost' k organicheskim soedineniiam olova.

Resistance to organic tin compounds of P. aeruginosa and E. coli carrying antibiotic resistance plasmids and to P. putida containing biodegradation plasmids was studied. It was shown that 5 resistance plasmids and the biodegradation CAM plasmid of Pseudomonas increased 3-4 times the strain resistance to triethylstannylsuccinylimide and triethylstannylmaleinimide. All these plasmids belong to the P-2 incompatibility group and also determine the bacterial resistance to potassium tellurite. Isolation and investigation of the mutant plasmids loosing simultaneously the capacity for determination of resistance to potassium tellurite and organic tin compounds suggest that resistance to these compounds in the investigated plasmids is determined by the same genetic system.