Abstract：
:We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Friedman AL,Geeves MA,Manstein DJ,Spudich JAdoi
10.1021/bi973143fsubject
Has Abstractpub_date
1998-07-07 00:00:00pages
9679-87issue
27eissn
0006-2960issn
1520-4995pii
bi973143fjournal_volume
37pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract：:S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requir...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi900590m
更新日期：2009-07-14 00:00:00
abstract：:Two terminally blocked peptides, acetylAETAAAKFLRQHMamide and acetylAETSSSRYLRQHMamide, were obtained by solid-phase synthesis, purified by reversed-phase chromatography, and characterized by fast atom bombardment mass spectrometry. Both peptides were soluble in aqueous solutions and remained monomeric over the concen...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00427a048
更新日期：1989-01-10 00:00:00
abstract：:With knowledge of individual transfer free energies of chemical groups that become newly exposed on protein denaturation and assuming the group transfer free energy contributions are additive, it should be possible to predict the stability of a protein in the presence of denaturant. Unfortunately, several unresolved i...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi035908r
更新日期：2004-02-10 00:00:00
abstract：:Transfection of cos-1 cells with either prostaglandin endoperoxide H synthase-1 (PGHS-1) or -2 (PGHS-2) results in a mixed population of cells containing a diverse range of expressed enzyme. The use of fluorescent substrates and antibodies, in conjunction with fluorescence microscopy, provides the means to quantitate ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00021a034
更新日期：1995-05-30 00:00:00
abstract：:Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0506730
更新日期：2005-10-04 00:00:00
abstract：:In contrast to all other known pyrophosphatases, Moorella thermoacetica pyrophosphatase (mtCBS-PPase) contains nucleotide-binding CBS domains and is thus strongly regulated by adenine nucleotides. Stopped-flow measurements using a fluorescent AMP analogue, 2'(3')-O-(N-methylanthranoyl)-AMP (Mant-AMP), reveal that nucl...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi9019737
更新日期：2010-02-09 00:00:00
abstract：:From the larger set of 191 variants at all the variable contact positions in the turkey ovomucoid third domain, we selected a subset that consists of Asp, Glu, His, and Lys residues at eight of the nine contiguous P6-P3' positions (residues 13-21), the exception being P3-Cys16 which is involved in a conserved disulfid...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0269512
更新日期：2003-03-18 00:00:00
abstract：:G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., Mc...
journal_title：Biochemistry
pub_type： 杂志文章
doi：
更新日期：2000-08-01 00:00:00
abstract：:Isoform 1 of the mammalian Na(+)/H(+) exchanger (NHE1) is a ubiquitously expressed plasma membrane pH regulatory protein. It removes one intracellular H(+) in exchange for one extracellular Na(+). The 500 N-terminal amino acids comprise the catalytic membrane domain and fold into 12 transmembrane (TM) segments. To gai...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi500392y
更新日期：2014-06-10 00:00:00
abstract：:To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[S...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi970608t
更新日期：1997-07-15 00:00:00
abstract：:Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution. The structure reveals a network of protein interact...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0601858
更新日期：2006-06-20 00:00:00
abstract：:F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi034746w
更新日期：2003-10-07 00:00:00
abstract：:We extended the technique of frequency-domain fluorometry to an upper frequency limit of 2000 MHz. This was accomplished by using the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity-dumped dye laser. We used a microchannel plate photomultiplier as the detector to obtain ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00375a012
更新日期：1987-01-13 00:00:00
abstract：:Western blot analysis, using a polyclonal antibody to the 240-kDa endogenous inhibitor of the 20 S proteasome, revealed that the inhibitor is a component of the 26 S complex. Although isolated inhibitor displayed a single 40-kDa band on SDS-PAGE, the antibody detected a 55-kDa component in the 26 S proteasome complex....
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00163a001
更新日期：1992-12-08 00:00:00
abstract：:The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi020462k
更新日期：2003-03-04 00:00:00
abstract：:The diazomethyl ketones of z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a m...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00645a033
更新日期：1977-12-27 00:00:00
abstract：:PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], w...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi047539k
更新日期：2005-09-13 00:00:00
abstract：:Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroup...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00436a017
更新日期：1989-05-16 00:00:00
abstract：:Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depen...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00474a018
更新日期：1990-06-05 00:00:00
abstract：:Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi050131i
更新日期：2005-06-21 00:00:00
abstract：:Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfo...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi901034r
更新日期：2009-10-13 00:00:00
abstract：:β-Lactam antibiotics have faced obsolescence with the emergence of methicillin-resistant Staphylococcus aureus (MRSA). A complex set of events ensues upon exposure of MRSA to these antibiotics, which culminates in proteolysis of BlaI or MecI, two gene repressors, and results in the induction of resistance. We report s...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi500074w
更新日期：2014-03-18 00:00:00
abstract：:The homeodomain proteins a1 and alpha 2 act cooperatively to regulate cell type specific genes in yeast. The basis of the cooperativity is a weak interaction between the two proteins which forms heterodimers that bind DNA tightly and specifically. In this paper, we examine the mechanism of heterodimerization. We show ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00197a033
更新日期：1994-08-09 00:00:00
abstract：:Calmodulin forms a covalent, one to one, complex with 3H-labeled norchlorpromazine isothiocyanate. Complex formation was monitored by high-performance liquid chromatography using a CN reverse-phase column which resolves calmodulin, the calmodulin-norchlorpromazine adduct, and norchlorpromazine isothiocyanate. Formatio...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00293a003
更新日期：1983-11-22 00:00:00
abstract：:Fluorescence lifetimes and fluorescence anisotropy decays of the voltage-sensitive styryl-pyridinium dye RH421 have been measured in the presence of dimyristoylphosphatidylcholine vesicles, the water soluble enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and Na+,K(+)-ATPase-containing membrane fragm...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00037a015
更新日期：1995-09-19 00:00:00
abstract：:The p53 protein family is involved in the control of an intricate network of genes implicated in cell cycle, through to germ line integrity and development. Although the role of p53 is well-established, the intrinsic nature of its homologue p73 has yet to be fully elucidated. Here, the biochemical characterization and...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi7023207
更新日期：2008-03-11 00:00:00
abstract：:Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-222...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00149a036
更新日期：1992-09-01 00:00:00
abstract：:Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecula...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00467a022
更新日期：1990-04-17 00:00:00
abstract：:Triple-helical DNA shows increasing potential for applications in the control of gene expression (including therapeutics) and the development of sequence-specific DNA-cleaving agents. The major limitation in this technology has been the requirement of homopurine sequences for triplex formation. We describe a simple ap...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00117a002
更新日期：1992-01-21 00:00:00
abstract：:The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00463a003
更新日期：1990-03-20 00:00:00
