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D D Hackney

Publications and source records attributed to D D Hackney.

At least 37 records · Page 2Linked to original sources
PubMed1992

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.

Animals↗
PubMed1992

Biochemical characterization of Escherichia coli DNA helicase I.

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single-stranded DNA-stimulated ATPase rate indicates that there is co-operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O-exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low-level reversal of the hydrolytic step during each turnover.

Adenosine Triphosphatases↗
PubMed1991

Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin.

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.

Adenosine Triphosphatases↗
PubMed1991

The mechanism of ATP hydrolysis by smooth muscle myosin and subfragments using steady state titration and 18O exchange.

The mechanisms of increases in the ATPase rates of smooth muscle acto-myosin, acto-heavy meromyosin (HMM) and acto-subfragment 1 (S1) were investigated using steady state titration and 18O exchange. Phosphorylation increased the phosphate release rates both from acto-myosin and acto-HMM. Steady state titration at high enzyme concentrations and 18O exchange at substoichiometric ATP concentrations showed that gizzard myosin was kinetically homogeneous, whereas HMM and S1 prepared by various published methods were heterogeneous. At high ATP concentrations, a small population of HMM and S1 hydrolyzed ATP with a low amount of oxygen exchange.

Adenosine Triphosphate↗
PubMed1989

Nucleotide-free kinesin hydrolyzes ATP with burst kinetics.

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.

Adenosine Triphosphatases↗
PubMed1988

Kinesin ATPase: rate-limiting ADP release.

The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from approximately equal to 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki less than 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.

Adenosine Triphosphatases↗
PubMed1987

Analysis of positional isotope exchange in ATP by cleavage of the beta P-O gamma P bond. Demonstration of negligible positional isotope exchange by myosin.

A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium with HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the beta P-O gamma P bond. This cleavage yields Pi derived from the gamma-phosphoryl of ATP that contains all four of the gamma oxygens. Both PIX between the beta,gamma-bridge and beta-nonbridge positions and washout of the gamma-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the beta-nonbridge positions and 18O at the beta,gamma-bridge and gamma-nonbridge positions. Application of this method to ATP in equilibrium with HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of gamma-nonbridge oxygens in the virtual absence of PIX. At 25 degrees C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the beta-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 degrees C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., & Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

Adenosine Triphosphate↗
PubMed1987

A residence-time analysis of enzyme kinetics.

A 'first-passage-time' analysis is applied to enzyme kinetics. It is shown that the residence times determined in this way are directly related to the steady-state parameters and are particularly useful in analysis of isotopic exchange. A simple linear means is used for the calculation of these residence times that makes this method easily applicable to the numerical evaluation of complex models. This stochastic type of approach provides an alternative that avoids the classical steady-state approximation that the concentrations of enzyme intermediates are constant. Instead, steady state is defined as the randomization of the states of the enzyme following initial mixing due to completion of the turnovers of individual enzyme molecules at different times.

Enzymes↗
PubMed1986

Anomalous oxygen-18 exchange during ATP synthesis in oxidative phosphorylation.

The synthesis of ATP from highly enriched [18O]Pi by submitochondrial particles driven by succinate oxidation produces distributions of 18O-labeled ATP species that deviate from the distributions predicted by a simple model for the exchange. Control experiments indicate no change in isotopic distribution when [18O]ATP is synthesized from [18O]ADP by adenylate kinase, which is bound to the submitochondrial particles. The observed deviations are in the opposite direction from that produced by heterogeneity due to multiple pathways for ATP synthesis. Two types of complex models can account for the observed deviations. One model has nonequivalence of the Pi oxygens during the exchange reaction, due to incomplete randomization of the Pi oxygens during the reversible cycles of hydrolysis and synthesis of bound ATP. The other model assumes that, during each turnover, a slow transition must occur between a high-exchange and a low-exchange pathway.

Adenosine Triphosphate↗
PubMed1985

Steady state kinetics at high enzyme concentration. The myosin MgATPase.

The rate of ATP hydrolysis by myosin at high concentrations with an ATP-regenerating system increases linearly with increasing added ATP up to a sharp break at the equivalence point of 1 ATP/myosin active site. Theoretical modeling indicates that the data require a KM on the order of the 30 nM value predicted by the rapid kinetic work (Lymn, R. W., and Taylor, E. W. (1970) Biochemistry 7, 2975-2983). Changes in the experimental conditions are found to change the slope of the initial increase in ATPase rate, but not to change the equivalence point. Proteolytic subfragments of myosin do not exhibit a linear initial increase in rate indicating that they are not homogeneous. Purified myosin is also found to show a small additional increase in ATPase rate at much higher ATP levels with a corresponding increase in flux through a pathway with a low extent of oxygen exchange. This high Km component with low oxygen exchange is distinct from the contaminating ATPase reported previously (Sleep, J. A., Hackney, D. D., and Boyer, P. D. (1980) J. Biol. Chem. 255, 4094-4099) which is shown here to be the CaATPase of the sarcoplasmic reticulum.

Adenosine Triphosphatases↗
PubMed1984

Catalytic consequences of oligomeric organization: kinetic evidence for "tethered" acto-heavy meromyosin at low ATP concentrations.

The influence of the supramolecular organization of myosin on its ATPase activity was investigated at a range of ATP concentrations, using as a model system subfragment 1 (S1) and heavy meromyosin (HMM), which are respectively monomeric and dimeric proteolytic fragments of myosin. At low ATP levels in the presence of a molar excess of actin, dimeric HMM showed an increased rate of ATP hydrolysis relative to that for monomeric S1. This increased ATPase for HMM was inhibited by high concentrations of ATP, which reduced the acto-HMM ATPase rate to the lower level of acto-S1. This observation is consistent with the rapid ATP hydrolysis of acto-HMM at low ATP being due to rapid product release from a "tethered" acto-HMM species, which has product bound to one head group while the other head group remains bound to actin. At high concentrations of ATP, ATP binds to both head groups, resulting in net dissociation of HMM from actin. This model is supported by 18O exchange data. Acto-HMM hydrolyzed ATP with extensive exchange of water oxygens into Pi at high ATP levels, but not at low ATP levels. Acto-S1 exhibited extensive exchange at both high and low ATP levels. This result is consistent with rapid product release from a tethered acto-HMM intermediate at low ATP.

Adenosine Triphosphate↗
PubMed1982

Nonlinear dependence on actin of acto-heavy meromyosin 18O exchange and ATPase rate at low actin concentrations.

Hydrolysis of highly enriched [gamma-18O]ATP in unlabeled water by acto-heavy meromyosin at low actin concentration was found to be heterogeneous in that significant amounts of both the species containing 0 or 3 18O-labeled oxygens/phosphate were formed. Detailed quantitative comparison with theoretical distributions over a wide range of actin concentrations, however, indicated that the pathway which catalyzed ATP hydrolysis with a low extent of exchange only made a significant contribution at a low actin concentration and did not represent a major fraction of the total hydrolysis seen at higher actin concentrations. This low exchange component was also detected in the dependence on actin of the steady state ATPase. At low actin the steady state ATPase rate increased more rapidly as a function of actin concentration than predicted by the Km and Vmax for actin activation observed at moderate to high actin levels. This extra ATP hydrolysis at low actin correlates with that predicted for the low exchange pathway both with respect to the fraction of the ATP hydrolyzed and to its dependence on the actin concentration.

Actins↗
PubMed1980

Theoretical analysis of distribution of [18O]Pi species during exchange with water. Application to exchanges catalyzed by yeast inorganic pyrophosphatase.

A theoretical analysis has been derived which allows the analytical calculation of the complete distribution of 18O-labeled Pi species expected to occur during medium Pi equilibrium HOH exchange of [18O]Pi and to be produced by intermediate Pi equilibrium HOH exchange during net hydrolysis of [18O]PPi or other labeled phosphate compounds. The observed distributions with catalysis by yeast inorganic pyrophosphatase are found to agree closely with the theoretical values indicating that the exchange reaction can be adequately described by a unique value of the partitioning of bound Pi between release from the enzyme versus formation of bound PPi with loss of an oxygen to the water. The limitations on the exclusion of other mechanisms are discussed. The extent of this partitioning does change, however, under some experimental conditions. At low pH, with activation by Mg2+ or Mn2+, the relative rate of release of Pi is found to increase. The extent of exchange is also dependent on the nature of the activating metal, being greatest with Co2+. During PPi hydrolysis with PPi in excess over Mg2+, a shift to lower extents of exchange is observed.

Hydrogen-Ion Concentration↗