ATP hydrolysis

Abstract: We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochromec promote APAF-1 oligomerization, forming a large multimeric APAF-1·cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3.

Abstract: The hydrolysis of ATP by acto-HMM has been studied during the transient state using a rapid-mixing apparatus. The rate of substrate binding was slightly slower and the rate of hydrolysis of the first molecule of ATP was essentially the same as for heavy meromyosin (HMM) alone. The rate of acto-HMM dissociation after binding substrate was too fast to measure in a stopped-flow apparatus, consequently dissociation occurs before hydrolysis of the bound ATP. By U nder physiological conditions, namely, 0.1-0.15 M KC1 and greater than 1 mM MgC12, myosin ATPase activity is strongly inhibited. Natural or synthetic actomyosin under these same conditions exists as a precipitate. Addition of ATP usually leads to “clearing,” a decrease in turbidity of the system, which is indicative of dissociation of actin and myosin, at least as measured by physical methods such a flow birefringence or viscosity (Maruyama and Gergely, 1962). The ATPase activity in the clear phase is only slightly higher than that of myosin ATPase. As long as traces of Ca ions are present this phase is followed by precipitation and superprecipitation and a 10to 20-fold increase in ATPase activity. Although this activation is clearly related to the mechanism of contraction, a heterogeneous system is unsuitable for kinetic studies. A better understanding is obtained from studies of the heavy meromyosin-actin complex which remains essentially homogeneous and does not superprecipitate. The fundamental work of Eisenberg and Moos (1968, 1970; Eisenberg et al., 1969) has considerably clarified our understanding of this system. Two competing reactions are involved, the dissociation of actomyosin by ATP and the activation of myosin ATPase by actin. Analysis of the steady-state kinetics by Eisenberg and Moos showed that the behavior could be interpreted as a decrease in the HMM-actin association constant when ATP is bound and a large increase in actin-HMM ATPase relative to myosin. By interpreting the kinetics by a modified MichaelesMenten scheme, the corresponding association and steadystate rate constants were obtained by extrapolating to infinite actin concentration. The real rate of the activated enzyme was found to be about 200 times larger than for myosin. The scheme could be formulated as 20 seo-1 AM + A T P e A . M . A T P A M + A D P + P +AI 1 A 0.1 sec-1 M + A T P e M , A T P A M + ADP + P * From the Department of Biophysics, University of Chicago, Chicago, Illinois 60637. ReceiuedJuly 8, 1971. This work was supported by National Institutes of Health, Grant No. G M 1992, Muscular Dystrophy Association, and Life Insurance Medical Research Fund. E. W. T. acknowledges a Research Career Development award from the U. S . Public Health Service; R. W. L. acknowledges support from the U. S. Public Health Service Training Grant G M 780. t To whom to address correspondence, means of a rapid column separation procedure it was shown that actin combines with the myosin.ADP. P complex and displaces the products of the hydrolysis reaction. It is concluded that this step is responsible for activation of myosin ATPase by actin. A simple kinetic scheme which accounts for the transient and steady-state behavior is presented and compared with the contraction cycle postulated for the sliding filament mechanism. In this scheme hydrolysis occurs largely through the AM * ATP state, Recent studies on the transient state of ATP hydrolysis by myosin and HMM (Lymn and Taylor, 1970) have shown that the first mole of ATP is hydrolyzed at a rapid rate, roughly 100 sec-1, which is much faster than the maximum rate in the Eisenberg and Moos mechanism under comparable conditions. The slow steady-state rate for myosin ATPase was attributed to the rate-limiting dissociation of the products, ADP, and phosphate from the enzyme (Taylor et al., 1970). These studies therefore suggested a modified view of the actin activation mechanism in which actin might affect the rate of product dissociation rather than the hydrolytic step itself. The work presented here describes a study of the HMMactin system in the transient state. A provisional and no doubt oversimplified mechanism is proposed which is consistent with the transient state experiments as well as the steady-state studies of Eisenberg and Moos. The essential feastures of the mechanism are that actomyosin dissociation preceeds hydrolysis, and activation is produced by recombination of actin with the myosin-products complex with displacement of products. The steps in the mechanism can be identified with the contractile cycle postulated in the sliding filament model (Huxley, 1968; Pringle, 1967). Materials and Methods Proteins. Rabbit myosin and heavy meromyosin (HMM)’ were prepared as described previously (Finlayson et al., 1969). Acetone powder was prepared by the method of Tonomura and Yoshimura (1962) and actin was extracted from the powder at 0” by the method of Carsten and Mommaerts (1963). Concentrations of HMM and actin were determined using the difference in absorbance at 291 and 350 mp in 0.5 N NaOH ( e 2 5 1 €360 = 776 cm2/g for HMM; eZg1 €350 = 1150 cm2/g for actin). These figures were obtained by micro-Kjeldahl analyses assuming a nitrogen content of 16.7%:. For the calculations in this paper the molecular weights of HMM and actin are assumed to be 350,000 and 50,000, respectively. ATPase Measurements. The early phase of ATP hydrolysis was measured by determination of [ 32P]phosphate libera1 Abbreviation used is: HMM, heavy meromyosin. B I O C H E M I S T R Y , V O L . 1 0 , N O . 2 5 , 1 9 7 1 4617 L Y M N A N D T A Y L O R column, a three-way stop cock was turned to allow fluid flow from a buffer reservoir driven by a Harvard apparatus pump. The column and buffer were thermostated by circulating water from a temperature bath. Resolution was somewhat poorer than with the technique previously employed of successive layering on top of the resin bed but was adequate for separation of the enzyme-product complex and permitted successive mixing of substrate and actin with the myosin solution.

Abstract: Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

Abstract: ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved

Abstract: The citrate cleavage enzyme (EC 4.1.3.8) of rat liver is inhibited by adenosine diphosphate, which appears to compete with adenosine triphosphate. This effect may ensure that fatty acids are produced only when the ATP level is high. The "energy charge" of the adenylate system, defined as (ATP + ½ ADP)/(AMP + ADP + ATP), is proposed as a fundamental metabolic control parameter. Enzymes that utilize ATP and are inhibited by ADP or AMP will yield steep curves of velocity as a function of energy charge (resembling the steep curves of velocity as a function of substrate concentration that are characteristic of many regulatory enzymes) even in the absence of multiple sites and cooperative binding.