PubMed Health⌕ Search

Biomedical subjects

D R Trentham

Publications and source records attributed to D R Trentham.

At least 73 records · Page 4Linked to original sources
PubMed1982

Relaxation of muscle fibres by photolysis of caged ATP.

A novel method has been developed for studying the reaction kinetics of the force-generating mechanism in muscle. Inert photolabile precursors of ATP or ADP are incorporated into muscle fibres having their surface membrane barrier removed. The nucleotide is then rapidly liberated by laser pulse photolysis. This circumvents the limitation in time resolution set by diffusion of nucleotide from the medium bathing the fibre. This laser photolysis method may be applicable to studies of the dynamic properties of many biological systems.

Adenosine Triphosphate↗
PubMed1982

Protein-bound adenosine 5'-triphosphate: properties of a key intermediate of the magnesium-dependent subfragment 1 adenosinetriphosphatase from rabbit skeletal muscle.

The time course of formation and decay of protein-bound adenosine 5'-triphosphate (ATP) has been monitored during single turnovers of the myosin subfragment 1 ATPase with nonspectrophotometric techniques. The rate constant controlling the ATP cleavage step increases markedly with ionic strength, so that in low salt the protein--ATP complex is observed transiently at higher concentration than the protein-products complex. The kinetics of the ATP cleavage step in a single turnover of the actosubfragment 1 ATPase indicates that under appropriate conditions this step is partially rate limiting during overall steady-state ATPase activity. It follows that a binary subfragment 1-ATP complex is a significant component of the steady-state intermediate of the actosubfragment 1 ATPase. Transient kinetic studies of ATP and adenosine 5'-(3-thiotriphosphate) [ATP (gamma S)] binding show directly that a substrate-induced protein isomerization accompanies ligand binding. The rate constant of the isomerization is 170 s-1 at pH 7.0, 15 degrees C, and 0.01 M ionic strength. Under these conditions nucleotide binding appears to be accompanied by a protein fluorescence increase that is 50% of the increase associated with magnesium-dependent steady-state ATPase activity.

Adenosine Triphosphatases↗
PubMed1982

Electron paramagnetic resonance studies of MN(II) complexes with myosin subfragment 1 and oxygen 17-labeled ligands.

Ligands in the first coordination sphere of Mn(II) in the complex of MnADP with myosin subfragment 1 from rabbit skeletal muscle have been investigated. EPR spectroscopy was used to detect superhyperfine coupling between unpaired electrons of the metal ion and the nuclei of oxygen atoms specifically labeled with oxygen 17. The results show that ADP is a beta-monodentate ligand for Mn(II) and that there are probably two water oxygens directly bound to Mn(II). The inhibitory complex of vanadate with subfragment 1 . MnADP was also investigated. Vanadate-dependent changes in the EPR spectra for enzyme-bound Mn(II) indicate that the coordination sphere of MN(II) changes upon binding of vanadate. ADP remains a beta-monodenate ligand in the complex and experiments with 17O-labeled water indicate that two oxygen atoms originally in water are ligands in the complex. However, the oxygens of vanadate equilibrate with those of water during sample preparation so that one of these ligands may be a vanadate oxygen. Three additional ligands, probably from the protein, are required to complete the sextet of ligands to Mn(II) in both complexes studied.

Adenosine Diphosphate↗
PubMed1981

The mechanism of ATP hydrolysis catalyzed by myosin and actomyosin, using rapid reaction techniques to study oxygen exchange.

During ATP hydrolysis, myosin or subfragment 1 catalyzes the exchange of oxygens between water and phosphate, so that on average, each product Pi molecule contains more than one oxygen atom derived from water. Using quenched-flow techniques, the exchange process in both ATP and Pi was studied in the rabbit skeletal muscle subfragment 1 ATPase. The exchange in protein-bound ATP (M*.ATP) and protein-bound ADP.Pi (M**.ADP.Pi) was followed as a function of time. The pattern of exchange follows closely a model in which M*.ATP + H2O and M**.ADP.Pi are interconverted directly, without any characterizable intermediate between these species. All oxygen atoms of Pi are equivalent with respect to loss to solvent, and elimination of water to re-form ATP occurs with a rate constant of 15 s-1 (at pH 8.0, I 0.015 M, 20 degrees C). This is the first direct measurement of the rate constant for the transformation of M**.ADP.Pi to M*.ATP. Oxygen exchange during steady state ATP hydrolysis in the presence of acto-subfragment 1 also fits well to this model, with actin reducing the time available for M*.ATP and M**.ADP.Pi to undergo exchange.

Actomyosin↗
PubMed1981

The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase.

The stereochemical course of the phosphoric residue transfer from ADP to water catalyzed by the (Mg2+ + Ca2+)-dependent ATPase of sarcoplasmic reticulum has been determined. For this determination, the preparation is described of ATP gamma S, stereospecifically labeled in the gamma-position with both 17O and 18O. After hydrolysis of this nucleotide, the analysis of the product inorganic [16O,17O,18O]thiophosphate showed that the reaction proceeded with retention of configuration at the gamma-phosphorus atom. This result is expected since a phosphoenzyme is well characterized for this ATPase and provides support for the hypothesis that each phosphate transfer step occurs with inversion. In this case, the formation and breakdown of the phosphoenzyme occur each with inversion leading to the retention observed for the whole reaction.

Adenosine Triphosphate↗
PubMed1980

The stereochemical course of phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase.

The stereochemical course of phosphoric residue transfer has been determined for beef heart mitochondrial ATPase. When aden 5'-(3-thiotriphosphate), stereospecifically labeled with 18O in the gamma position, was hydrolyzed in [17O]water in the presence of the ATPase, the product inorganic [16O, 17O, 18O]thiophosphate was chiral. The configuration of the product showed that the hydrolysis had proceeded with inversion at the gamma-phosphorus atom. This result suggests that there is a direct, in-line transfer of the phosphoric residue between ADP and water and that there is no phosphoenzyme intermediate.

Adenosine Triphosphatases↗
PubMed1980

Mechanism of adenosine 5'-triphosphate cleavage by myosin: studies with oxygen-18-labeled adenosine 5'-triphosphate.

During the hydrolysis of MgATP catalyzed by myosin, ATP bound to the protein undergoes a reaction such that the beta-nonbridge oxygen atoms exchange position with the beta gamma-bridge oxygen atom. The extent of this exchange was variable but averaged 45% for ATP that had been bound for 2 s at the myosin subfragment 1 active site at ionic strength 0.08 M, pH 8.0, and 22 degrees C. This result proves that ATP cleavage in the myosin active site is readily reversible. The result also suggests that the beta-phosphate of ADP that must be formed in this cleavage step is highly constrained in the protein.

Adenosine Triphosphatases↗
PubMed1980

The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction.

When adenosine 5'-(3-thiotriphosphate), stereospecifically labeled in the gamma position with 18O, was hydrolyzed in the presence of myosin subfragment 1 in 17O-enriched water, the product inorganic [16O,17O,18O]thiophosphate was chiral. The configuration of this product showed that the hydrolysis proceeds with inversion at the transferred phosphoric residue. This result suggests a direct, in-line hydrolysis mechanism for the ATPase.

Adenosine Triphosphatases↗
PubMed1980

Analysis of chiral inorganic [16O, 17O, 18O]thiophosphate and the stereochemistry of the 3-phosphoglycerate kinase reaction.

The R- and S-enantiomers of inorganic [16O, 17O, 18O]-thiophosphate have been synthesized from the B and A isomers of [alpha-S; alph-18O; alpha beta-17O; beta-17O3]ADP. Each chiral thiophosphate was incorporated into isomer A of ATP beta S with distinct oxygen isotopic labelings, which were characterized by 31P NMR. The 3-phosphoglycerate kinase-catalyzed reaction was shown to proceed by inversion at phosphorus since all other steps in the reaction sequence between inorganic thiophosphate and ATP beta S were of known stereospecificity.

Magnetic Resonance Spectroscopy↗
PubMed1980

A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1-(2-nitro phenylethyladenosine 5'-triphosphate "caged ATP" was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 microM ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 microM ATP was found to be controlled by a process having a rate constant of 2.2 X 10(9) [H+] sec-1 at 22 degrees C at pH 5.8-9.5, which corresponds to 220 sec-1 at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds.

Actomyosin↗
PubMed1979

Magnesium ion dependent rabbit skeletal muscle myosin guanosine and thioguanosine triphosphatase mechanism and a novel guanosine diphosphatase reaction.

The mechanism of the Mg2+-dependent myosin subfragment 1 catalyzed hydrolysis of GTP and 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (thioGTP) has been investigated by rapid-reaction techniques. The myosin was isolated from rabbit skeletal muscle. The steady-state intermediate of these reactions consists pre-dominantly of a protein-substrate complex unlike the myosin subfragment 1 ATPase reaction which has a protein-products complex as the principal steady-state component. The mechanism of GTP hydrolysis catalyzed by subfragment 1 has other marked differences from the ATPase mechanism. The second-order rate constant of binding of GTP to subfragment 1 is tenfold greater than that for GDP binding. The dissociation rate constant of GDP from subfragment 1 is 0.06 s-1 compared with the subfragment 1 catalytic center activity for GTP hydrolysis of 0.5 s-1 at pH 8.0 and 20 degrees C. This shows that GDP bound to subfragment 1 forms a complex which is not kinetically competent to be an intermediate of the GTPase mechanism. GDP is hydrolyzed in the presence of subfragment 1 to GMP and Pi. The subfragment 1 GTPase mechanism has a nuber if features in common with that of the elongation factor Tu GTPase of the protein biosynthetic system of Escherichia coli.

Animals↗
PubMed1978

Kinetics of oxygen-18 exchange between inorganic phosphate and water catalyzed by myosin subfragment 1, using the 18O shift in 31P NMR.

The time course of oxygen-18 exchange between [18O]Pi and normal water, catalyzed by myosin subfragment 1 in the presence of MgADP, was followed using the shift in 31P NMR caused by the presence of oxygen-18 bound to the phosphorus. Essentially all molecules of [18O]Pi that bind to the enzyme undergo complete exchange and are released as [16O4]Pi. Exchange probably occurs by formation of myosin.ATP from a myosin.ADP.Pi complex and is rapid relative to release of Pi from this complex. The kinetics of exchange give a value for the rate constant for binding Pi to myosin.ADP of 0.23 M-1 S-1 (pH 8.0, 22 degrees C). This value is consistent with exchange occurring by reversal of the ATP-ase reaction back to the myosin.ATP complex.

Animals↗
PubMed1978

Preparation and characterization of frog muscle myosin subfragment 1 and actin.

The preparation, structural and steady-state kinetic characteristics of contractile proteins from the leg muscle of frogs Rana temporaria and Rana pipiens are described. Actin and myosin from the two frog species are indistinguishable. The proteins have structural and steady-state kinetic properties similar to those from rabbit fast-twitch skeletal muscle. Chymotrypsin digestion of frog myosin or myofibrils in the presence of EDTA yields subfragment 1, which is separated by chromatography into two components that are distinguished by their alkali light-chain content.

Actins↗
PubMed1978

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.

Actins↗
PubMed1977

The interaction of chromophoric nucleotides with subfragment 1 of myosin.

The interaction of a series of chromophoric nucleotides derived from 6-mercapto-9-beta-ribofuranosylpurine (thioinosine, thiol) and 2-amino-6-mercapto-9-beta-ribofuranosyl-purine (thioguanosine, thioG) with myosin subfragment 1 isolated from rabbit skeletal muscle was investigated kinetically and spectroscopically. The Mg2+-dependent hydrolyses of thioITP and thioGTP are catalysed by subfragment 1 and probably proceed by a similar mechanism as for ATP hydrolysis, although with different rate constants. For example, the binary thioGDP-protein complex only comprises 8% of the steady-state intermediate of the thioGTPase at 5 degrees C and pH 6.5. Long-lived analogues of intermediates of the thioGTPase were generated by using thioGTP(gammaS) [thioguanosine 5'-(3-thio)-triphosphate], thioGMP-P(NH)P (5'-thioguanylylimidodiphosphate) and thioGDP. The near-u.v. spectra of the thioguanosine nucleotides bound to subfragment 1 were measured and showed that in all cases the purine ring is bound to the protein in a hydrophobic environment, although the pK of the purine thiol group only increases by 0.2-0.3. ThioGTP caused glycerinated rabbit psoas muscle to contract, but in contrast with thioITP was not able to relax muscle. The applications of these chromophoric nucleotides for investigating the mechanism of muscle contraction and other biological systems, particularly those involving guanosine nucleotide regulation, are discussed.

Adenosine Triphosphate↗
PubMed1976

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme.

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

Chemical Phenomena↗