Lack of communication between LC2 light chain and the SH1 region of myosin S-1 studied by 19F-NMR

Myosin subfragment-1 (S-1) which contains the LC2 light chain has been labelled with fluorine to allow an 19F-NMR study of the coupling and energetics of structural changes in the myosin head. Two fluorine-containing reagents, N-4-(trifluoromethyl)phenyl iodoacetamide and N-3,5-di(trifluoromethyl)phenyl iodoacetamide, have been used to label the myosin heavy chain at the unusually reactive sulfhydryl-1 (SH1) position. The chemical shift of both reagents on S-1 is sensitive to a structural transition in the region of SH1 which occurs upon increasing the temperature from 0 degrees C to 35 degrees C. The midpoint of the transition in both papain and chymotryptic S-1 is at approximately 11 degrees C at pH 7 (0.1 M CKl). The temperature dependence of the chemical shift may be fit assuming a two-state equilibrium where delta G degree' (T) = 101-110T +0.386 T2 (where T is the temperature in Kelvin). Both delta H degree' (T) and delta S degree' (T) have a small temperature dependence from 0 to 35 degrees C: at 20 degrees C, delta H degree' (T) = -33 kcal/mol. delta S degree' (T) = -116 e.u. and delta Cp = -226 cal/mol per deg (pH 7.0, 0.1 M KCl). The NMR data indicate that the presence of the LC2 light chain in papain S-1 does not modify the structure of S-1 in the vicinity of SH1, nor does it modify the energetics of the structural transition from that seen in its absence with chymotryptic S-1. The presence of calcium which is bound by the LC2 of papain S-1 also does not alter the energetics of the transition. Thus it would appear that the LC2 light chain (on myosin S-1) does not participate in the two-state transition, nor does it interact strongly with regions of the heavy chain which participate in the transition.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Isocitrate dehydrogenase in D. melanogaster imaginal discs: pattern development and alteration by homoeotic mutant genes

The distribution of the soluble form of NADP+-dependent isocitrate dehydrogenase (ICDH) was examined in Drosophila melanogaster imaginal discs. Development of the enzyme patterns and the specific transformations of the patterns by homoeotic mutants were studied. ICDH pattern formation was followed in eye-antennal discs and wing discs from the late 2nd instar stage through 3rd instar and 8 hours into prepupal development. The patterns formed gradually in both disc types. The most interesting pattern developed in the eye portion of the eye-antennal disc complex. ICDH distribution as well as staining intensity correlated well with differentiation of the ommatidia. The spatial distribution of ICDH within the discs was under genetic control. The patterns reflected the state of determination of the disc. When the presumptive tissue type was transformed via mutant homoeotic genes to different determinative states, the ICDH pattern likewise transformed to the pattern characteristic of the newly acquired structure.     

Pyridoxal 5'-diphospho-5'-adenosine binds at a single site on isolated alpha-subunit from Escherichia coli F1-ATPase and specifically reacts with lysine 201

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated alpha-subunit and [3H] PLP-AMP after reduction with NaBH4. alpha-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide. Automated amino acid sequencing showed that residue alpha-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex. PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

Human cells infected with retrovirus vectors acquire an endogenous murine provirus.

A 5.2-kilobase mouse RNA is expressed in human cells following infection with recombinant retroviruses propagated in mouse NIH 3T3 cells as psi-2 pseudotypes. This RNA is transcribed from a defective mink cell focus-forming provirus and copackaged into virions and integrated into human target cell DNA at a frequency comparable to that of the recombinant retrovirus genome.     

Genetic mapping of a cloned sequence responsible for susceptibility to ecotropic murine leukemia viruses.

A mouse cDNA that confers susceptibility to ecotropic murine leukemia viruses following transfection into human EJ cells has been cloned and sequenced. We show that this sequence is likely to be Rec-1, the chromosome 5 locus originally defined by studies with somatic cell hybrids as responsible for virus susceptibility, and provide a specific chromosomal map position for this locus by analysis of an interspecies backcross. This locus maps in the distal region of chromosome 5 and is thus not within the cluster of retrovirus-related genes near the centromere.     

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.     

Comparison of twitch potentiation in the gastrocnemius of young and elderly men

The capacity for twitch potentiation in the gastrocnemius muscle was determined following maximal voluntary contractions (MVC) in 11 elderly (means +/- SD; 66.9 +/- 5.3 years) and 12 young (25.7 +/- 3.8 years) men. Potentiation was observed by applying selective stimulation to the muscle belly, 2 s after a 5 s MVC. With this procedure, both groups showed significant (P less than 0.05) increases in twitch tension in the gastrocnemius (ratios of potentiated twitch to baseline were means = 1.68 +/- 0.40 for young vs means = 1.40 +/- 0.20 for the elderly, P less than 0.001). Time to peak tension of the twitch decreased from means = 101.5 +/- 17.9 ms to means = 88.0 +/- 15.8 ms in the young men following potentiation; the respective values for the older men were 136.7 +/- 17.9 ms and 133.1 +/- 28.6 ms. These changes resulted in a greater rate of tension development in the potentiated state. The elderly gastrocnemius thus showed qualitatively similar changes in the isometric twitch following potentiation, but reduced and prolonged responses in comparison to young adults. Slowed muscle contraction and reduced capacity for potentiation may be physiological correlates of the reported morphological changes in aged skeletal muscle.     

Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers.

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.