Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Effect of actin, ATP, phosphates, and pH on vanadate-induced photocleavage of myosin subfragment 1.

Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K+(EDTA)-, Ca2(+)-, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of actin-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the "consensus" ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the binding and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the "consensus" site through a loop. The 74-kDa cleavage site is a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

The RNase PARN Controls the Levels of Specific miRNAs that Contribute to p53 Regulation

1. Download high-res image (126KB)
2. Download full-size image

     

Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.