Abstract:
:Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Stahl N,Jencks WPdoi
10.1021/bi00398a019subject
Has Abstractpub_date
1987-12-01 00:00:00pages
7654-67issue
24eissn
0006-2960issn
1520-4995journal_volume
26pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Chromaffin granules isolated from bovine adrenal medulla were labeled with small unilamellar vesicles (SUV) made from N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and/or N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine as donor and acceptor fluorophores. Labeling of granules could be followed...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00315a020
更新日期:1984-09-25 00:00:00
abstract::The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthe...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00939
更新日期:2019-04-30 00:00:00
abstract::Large primary deuterium kinetic isotope effects (1° DKIEs) on enzyme-catalyzed hydride transfer may be observed when the transferred hydride tunnels through the energy barrier. The following 1° DKIEs on kcat/ Km and relative reaction driving force are reported for wild-type and mutant glycerol-3-phosphate dehydrogenas...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00536
更新日期:2018-07-24 00:00:00
abstract::West Nile virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection can cause severe neurological disease and fatality in humans. Efforts are ongoing to develop antiviral drugs that inhibit the WNV protease, a viral enzyme required for polyprotein processing. Unfortunately, lit...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi400782w
更新日期:2013-10-01 00:00:00
abstract::The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specif...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi951975c
更新日期:1996-04-02 00:00:00
abstract::Seventeen ribosomal proteins of Saccharomyces cerevisiae were isolated from small ribosomal subunits and disodium ethylenediaminetetraacetate treated 80S ribosomes by chromatography on a column of carboxymethylcellulose and/or by filtration through Sephacryl S-200. The isolated proteins are YS4, YS7, YS8, YS9, YS10, Y...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00262a005
更新日期:1982-09-14 00:00:00
abstract::The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00690a030
更新日期:1975-09-23 00:00:00
abstract::It is now accepted that two or more conformations may exist within the same DNA molecule, thereby generating conformational junctions. The presence of B-Z junctions between right- and left-handed DNA conformations has been detected in plasmids by a number of techniques. Preliminary characterization of the first exampl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00428a046
更新日期:1989-01-24 00:00:00
abstract::We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan fluorescence,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00199a046
更新日期:1994-08-23 00:00:00
abstract::Alpha-synuclein (alpha-syn) is a 140-residue protein that aggregates in intraneuronal inclusions called Lewy bodies in Parkinson's disease (PD). It is composed of an N-terminal domain with a propensity to bind lipids and a C-terminal domain rich in acidic residues (the acidic tail). The objective of this study was to ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi060939i
更新日期:2006-09-12 00:00:00
abstract::Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002938a
更新日期:2001-08-07 00:00:00
abstract::Pig kidney general acyl-CoA dehydrogenases forms the blue neutral radical on dithionite or photochemical reduction (Thorpe, C., Matthews, R. G., & Williams, C. H. (1979) Biochemistry 18, 331-337] in accord with its classification as a flavoprotein dehydrogenase. However, dithionite reduction of the enzyme in the prese...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00520a024
更新日期:1981-08-18 00:00:00
abstract::We previously isolated a unique anticoagulant protein named IX/X-bp (factor IX/factor X-binding protein) from the venom of the habu snake Trimeresurus flavoviridis. We recently determined its primary structure and found that this protein had a structure homologous to the carbohydrate-recognition domains of C-type lect...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00078a012
更新日期:1993-07-13 00:00:00
abstract::Two antisera used in the radioimmunoassay for human fibrinopeptide A (FPA) which appear to have different immunochemical specificities have been tested for cross-reactivity with fibrinogen and with three fragments of fibrinogen which contain the FPA sequence. These fragments were the three-chain, NH2-terminal disulfid...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00651a004
更新日期:1976-03-23 00:00:00
abstract::Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4'-phosphopantetheine (4'-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4'-PP is mediated by 4' phosphopantetheinyl transfersases (PPTases). Mycobacter...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi501444e
更新日期:2015-04-14 00:00:00
abstract::The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the trypt...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00680a018
更新日期:1975-05-06 00:00:00
abstract::Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00148a034
更新日期:1992-08-25 00:00:00
abstract::The quercetin binding sites on spinach chloroplast coupling factor 1 (CF1) have been investigated using direct and competitive binding, stopped-flow, temperature-jump, and fluorescence resonance energy transfer measurements. It was found that 8-anilino-1-naphthalensulfonic acid (ANS) competes with quercetin binding at...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00646a001
更新日期:1976-01-13 00:00:00
abstract::Kinetic study of a series of compounds containing the thrombin-directed peptide D-Phe-ProboroArg-OH had indicated that the structure of the N-terminal blocking group may be correlated with binding [Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289-18297]. In order to further study this phenomeno...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi952164b
更新日期:1996-07-30 00:00:00
abstract::For the elucidation of the mechanism of steroid hormone receptor activation, the hydrodynamic properties of the unactivated and activated forms of the nonproteolyzed glucocorticoid receptor from the mouse AtT-20 pituitary tumor cell line were determined. The unactivated, molybdate-stabilized receptor has the following...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00277a038
更新日期:1983-04-12 00:00:00
abstract::Activation enthalpies and entropies are reported for proton-deuteron exchange at 42 amide sites in T4 lysozyme and compared with activation volumes for the same residues obtained earlier [Hitchens, T. K., and Bryant, R. G. (1998) Biochemistry 37, 5878-5887]. There is no correlation found between activation volume and ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991718y
更新日期:2000-01-11 00:00:00
abstract::We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00099a024
更新日期:1991-09-03 00:00:00
abstract::The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi992226o
更新日期:2000-03-07 00:00:00
abstract::Parkinson's disease (PD) involves loss of dopaminergic neurons in the substantia nigra and is characterized by intracellular inclusions, Lewy bodies, consisting primarily of aggregated alpha-synuclein. Two substitution mutations (A53T and A30P) in alpha-synuclein gene have been identified in familial early-onset PD. T...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi036114f
更新日期:2004-05-11 00:00:00
abstract::The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator pr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991303c
更新日期:1999-09-28 00:00:00
abstract::The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-depe...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00875
更新日期:2015-10-20 00:00:00
abstract::While calcium binding to troponin C (TnC) triggers the contraction of both skeletal and cardiac muscle, there is clear evidence that different mechanisms may be involved. For example, activation of heart myofilaments occurs with binding to a single regulatory site on TnC, whereas activation of fast skeletal myofilamen...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971223d
更新日期:1997-10-07 00:00:00
abstract::The interaction of solvent of the substrate binding domain of the bacterial heat shock 70 chaperone protein DnaK was studied in its apo form and with bound hydrophobic substrate peptide, using refined nuclear magnetic resonance experiments. Distinct differences between the two states of the protein were observed. Acco...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi030097c
更新日期:2003-09-30 00:00:00
abstract::These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, le...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002629k
更新日期:2001-06-12 00:00:00
abstract::The cytochrome P450-dependent terminal desaturation of valproic acid (VPA) is of both toxicological and mechanistic interest because the product, 4-ene-VPA, is a more potent hepatotoxin than the parent compound and its generation represents a rather novel metabolic reaction for the cytochrome P450 system. In the prese...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00024a013
更新日期:1995-06-20 00:00:00