The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Structural alterations and inhibition of unisite and multisite ATP hydrolysis in soluble mitochondrial F1 by guanidinium chloride

The effect of guanidinium chloride (GdnHCl) on the ATPase activity and structure of soluble mitochondrial F1 was studied. At high ATP concentrations, hydrolysis is carried by the three catalytic sites of F1; this reaction was strongly inhibited by GdnHCl concentrations of <50 mM. With substoichiometric ATP concentrations, hydrolysis is catalyzed exclusively by the site with the highest affinity. Under these conditions, ATP binding and hydrolysis took place with GdnHCl concentrations of >100 mM; albeit at the latter concentration, the rate of hydrolysis of bound ATP was lower. Similar results were obtained with urea, although nearly 10-fold higher concentrations were required to inhibit multisite hydrolysis. GdnHCl inhibited multisite ATPase activity by diminishing the V(max) of the reaction without significant alterations of the Km for MgATP. GdnHCl prevented the effect of excess ATP on hydrolysis of ATP that was already bound to the high-affinity catalytic site. With and without 100 mM GdnHCl and 100 microM [3H]ATP in the medium, F1 bound 1.6 and 2 adenine nucleotides per F1, respectively. The effect of GdnHCl on some structural features of F1 was also examined. GdnHCl at concentrations that inhibit multisite ATP hydrolysis did not affect the exposure of the cysteines of F1, nor its intrinsic fluorescence. With 100 mM GdnHCl, a concentration at which unisite ATP hydrolysis was still observed, 0.7 cysteine per F1 became solvent-exposed and small changes in its intrinsic fluorescence of F1 were detected. GdnHCl concentrations on the order of 500 mM were required to induce important decreases in intrinsic fluorescence. These changes accompanied inhibition of unisite ATP hydrolysis. The overall data indicate that increasing concentrations of GdnHCl bring about distinct and sequential alterations in the function and structure of F1. With respect to the function of F1, the results show that at low GdnHCl concentrations, only the high-affinity site expresses catalytic activity, and that inhibition of multisite catalysis is due to alterations in the transmission of events between catalytic sites.     

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.     

The complex of mitochondrial F1-ATPase with the natural inhibitor protein is unable to catalyze single-site ATP hydrolysis

Interaction of mitochondrial F₁-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [¹⁴C]nucleotides in F₁-ATPase during steady-state [¹⁴C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.     

The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 beta-subunit

The specific, mitochondrial ATP synthase protein (IF1) was covalently cross-linked to its binding site on the catalytic sector of the enzyme (F1-ATPase). The cross-linked complex was selectively cleaved, leaving IF1 intact to facilitate the subsequent purification of the F1 fragment to which IF1 was cross-linked. This fragment was identified by sequence analysis as comprising residues 394-459 on the F1 beta-subunit, near the C-terminus. This finding is discussed in the light of secondary structure predictions for both IF1 and the F1 beta-subunit, and sequence homologies between mitochondrial and other ATP synthases.     

Synthesis of ATP by soluble mitochondrial F1 ATPase and F1-inhibitor-protein complex in the presence of organic solvents

The F1 and F1-inhibitor-protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20-50% v/v), ethylene glycol (20-60% v/v) or poly(ethylene glycol) 4000 and 8000 (30-50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents. In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1-inhibitor-protein there was no synthesis of ATP in the absence of MgCl2. The rate of ATP synthesis became faster as the MgCl2 concentration in the medium was raised from 0.1-10 mM. The Km for Pi of F1 was in the range of 0.8-1.5 mM. The Km for Pi of the F1-inhibitor-protein was much higher than that of F1 and could not be measured. In the presence of 10 mM MgCl2 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1-inhibitor-protein were 5.2-10.4 h-1 and 3.5-5.9 h-1 respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h-1. The tightly bound ATP of F1 and F1-inhibitor-protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgCl2 concentration was suddenly decreased. Both in presence and absence of Mg2+, 40-60% of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non-radioactive ATP was added to the assay medium. This was not observed when F1-inhibitor-protein was used.     

Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase

A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.     

Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity

Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.     

Benzodiazepine-based selective inhibitors of mitochondrial F1F0 ATP hydrolase

A series of benzodiazepine-based inhibitors of mitochondrial F(1)F(0) ATP hydrolase were prepared and evaluated for their ability to selectively inhibit the enzyme in the forward direction. Compounds from this series showed excellent potency and selectivity for ATP hydrolase versus ATP synthase, suggesting a potentially beneficial profile useful for the treatment of ischemic heart disease.     

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis.

During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.     

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

tRNA-triggered ATP hydrolysis and generation of membrane potential by the leishmania mitochondrial tRNA import complex

Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNA(Tyr)(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F(1)-F(0)-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels.     

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500-650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Oscillatory accumulation of enzyme activity, enzyme protein and F1-inhibitor during the cell cycle.

1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.