Abstract:
:We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Forgacs E,Cartwright S,Kovács M,Sakamoto T,Sellers JR,Corrie JE,Webb MR,White HDdoi
10.1021/bi060712nsubject
Has Abstractpub_date
2006-10-31 00:00:00pages
13035-45issue
43eissn
0006-2960issn
1520-4995journal_volume
45pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core β-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00937
更新日期:2020-02-04 00:00:00
abstract::The active site of histidine decarboxylase (HDC) from Lactobacillus 30a contains a pyruvoyl cofactor sitting at the interface of two molecules in a trimer. Although exhibiting hyperbolic kinetics at pH 4.8, near its optimum, HDC is cooperative at pH 7.6, indicating that the units of the trimer communicate. A Hill plot...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00069a032
更新日期:1993-05-11 00:00:00
abstract::Complementary DNA (cDNA) clones encoding bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) have been isolated from a bovine testicular lambda gt11 library using polyclonal antibodies against 20 alpha-HSD and DNA probe hybridization. Nucleotide sequencing of three independently isolated clones was ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00057a003
更新日期:1993-02-16 00:00:00
abstract::Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00401a059
更新日期:1988-01-12 00:00:00
abstract::A novel single polypeptide endopeptidase of 24 kDa (24k-endopeptidase) was purified with a yield of 300-400 microg/L from conditioned medium of a bacterial strain which was identified as a new species in the genus Chryseobacterium Sp. on the basis of its 16S rDNA sequence and DNA:DNA hybridizations. The NH(2)-terminal...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi992014r
更新日期:2000-01-18 00:00:00
abstract::Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mon...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00247a013
更新日期:1991-08-20 00:00:00
abstract::The conformational change of the model peptide Ac-K-G-R-G-D-G-amide induced by a phospholipidic interface was investigated by proton nuclear magnetic resonance (1H NMR). In aqueous solution, the free peptide is highly flexible and disordered, even in the presence of deuterated dodecyl-phosphocholine (DPC-d38) micelles...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00124a018
更新日期:1992-03-10 00:00:00
abstract::MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0268807
更新日期:2003-03-25 00:00:00
abstract::Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00354a031
更新日期:1986-03-25 00:00:00
abstract::Modification of the bacterial cell wall heteropolymer peptidoglycan by addition of an acetyl group to the C-6 hydroxyl group of N-acetylmuramoyl residues is known to inhibit the activity of muramidases (lysozymes) of innate immune systems. The O-acetylation of peptidoglycan also precludes the action of intrinsic lytic...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051679s
更新日期:2006-01-24 00:00:00
abstract::Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetic...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00621a024
更新日期:1977-01-25 00:00:00
abstract::The circadian clock allows organisms to accurately predict the earth's rotation and modify their behavior as a result. Genetic analyses in a variety of organisms have defined a mechanism based largely on gene expression feedback loops. However, as we delve more deeply into the mechanisms of circadian timekeeping, we a...
journal_title:Biochemistry
pub_type: 杂志文章,评审
doi:10.1021/bi5008386
更新日期:2015-01-20 00:00:00
abstract::The extrinsic 33-kDa protein (P33) was cross-linked covalently to the binding site on P33-depleted PSII preparations which is responsible for reconstitution of photosynthetic water oxidation after PSII preparations have been washed with 1 M CaCl2. Conditions were found in which more than half of the cross-linked prote...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00439a033
更新日期:1989-06-27 00:00:00
abstract::The mechanism by which the heme-containing peroxidase, chloroperoxidase, is able to chlorinate substrates is poorly understood. One approach to advance our understanding of the mechanism of the enzyme is to determine those factors which contribute to its stability. In particular, under alkaline conditions, chloroperox...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961512m
更新日期:1996-11-19 00:00:00
abstract::Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0485878
更新日期:2004-12-14 00:00:00
abstract::Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi000808w
更新日期:2000-08-22 00:00:00
abstract::Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002080p
更新日期:2000-12-26 00:00:00
abstract::13C NMR has been used to demonstrate the metabolism of dilute solutions of labeled formaldehyde by Escherichia coli to methanol, formate, carbon dioxide, and several other unidentified metabolites which contain labeled CH2 groups. Aeration of bacterial suspensions within the spectrometer dramatically increased the rat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00298a017
更新日期:1984-01-31 00:00:00
abstract::The effects of site-directed mutagenesis were used to explore the role of residues in M4 on the apparent Ki of a selective, K+-competitive inhibitor of the gastric H+,K+ ATPase, SCH28080. A double transfection expression system is described, utilizing HEK293 cells and separate plasmids encoding the alpha and beta subu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991837d
更新日期:2000-03-21 00:00:00
abstract::In affinity selection, calcineurin selects from a population of randomly modified calmodulins those species with which it prefers to interact. The method shows that acetylation of lysines affects calmodulin so as to interfere with its ability to interact with calcineurin. Monoacetylation of any lysine of calmodulin re...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00379a026
更新日期:1987-03-10 00:00:00
abstract::The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistanc...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b01008
更新日期:2015-11-17 00:00:00
abstract::To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi100097f
更新日期:2010-06-15 00:00:00
abstract::Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00688
更新日期:2018-01-09 00:00:00
abstract::The three-dimensional high-resolution structure of rhodopsin is unknown, as is the case for almost all integral membrane patients. As part of an alternative approach to determine of membrane protein structure, we are pursuing the structure of cytoplasmic domains of this G-protein receptor. A peptide, rhoIII, with the ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00045a002
更新日期:1995-11-14 00:00:00
abstract::Fourier-transform infrared spectroscopy has been applied to the study of lipid vesicle-supported two-dimensional crystals and noncrystalline preparations of beef heart cytochrome oxidase. At room temperature, no conformational differences are seen between the noncrystalline and crystalline proteins, whose conformation...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00204a029
更新日期:1994-09-27 00:00:00
abstract::FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways i...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061763n
更新日期:2007-03-13 00:00:00
abstract::Glutathionylation of intracellular proteins is an established physiological regulator of protein function. In multiple models, including ischemia-reperfusion of the heart, increased oxidative stress results in the glutathionylation of sarcomeric actin. We hypothesized that actin glutathionylation may play a role in th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi900669m
更新日期:2009-08-11 00:00:00
abstract::The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family features several l-aminoacylases useful in biocatalysis. Mammalian Acy1, in particular, has been applied in racemic resolution and reverse hydrolysis. Despite recent advances in our understanding of the active site architecture and functioning, determinants of A...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi702156h
更新日期:2008-04-08 00:00:00
abstract::The betagamma-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca(2+) binding was found in a few betagamma-crystallin members earlier, although its prevalence and diversity as inherent molecular...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9017076
更新日期:2009-12-29 00:00:00
abstract::Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00635a033
更新日期:1977-08-09 00:00:00