ATP-generating systems

The a-oxoacid dehydrogenases yield CoA derivatives which may enter biosynthetic reactions. Alternatively, the acyl-CoA compounds may be cleaved with generation of ATP. The pyruvate formate-lyase system also operates as part of an ATP-generating system for anaerobic organisms, for example, in the "mixed acid fermentation" of enterobacteria such as E. coli (Chapter 17). These two reactions, which are compared in Fig. 15-16, constitute an important pair of processes both of which accomplish substrate-level phosphorylation. They should be compared with the previously considered examples of substrate level phosphorylation depicted in Eq. 14-23 and Fig. 15-16. 

Since ADP is inhibitory to the nitrogenase system, it is best in laboratory studies to supply ATP from an ATP-generating system such as a mixture of creatine phosphate, creatine kinase, and a small amount of ADP (Eqs. 6-65,... 

This process was extended to the phosphorylation of various substrates, in particular to the synthesis of ATP from ADP in mixed solvent [5.62a] and in aqueous solution in the presence of Mg2, probably via formation of a ternary catalytic species 83 [5.62b]. The latter abiotic ATP generating system has been coupled to sets of ATP consuming enzymes resulting in the production of NADH by a combined artificial/natural enzymatic process (Fig. 9) [5.63]. 

Fig. 9. Sequence of transformations catalysed by the supramolecular ATP-generating system [38, AcP, Mg2, ADP] (38 = [24]-N6C>2) and the enzymes hexokinase (HK), glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phospho-gluconate dehydrogenase (6-P-GDH) [5.32],...

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

The experimental design is simple. A given sample of nitrogenase equipped with an ATP-generating system, Mg2+, and reductant is allowed to turn over without substrate (case I), and the dihydrogen production is monitored. The amount of dihydrogen produced is found to be equal (within experimental error) to the dithionite oxidized (50). Therefore, the electron balance equation is ... 

The ATP-generating system of cells maintains the [ATP]/[ADP][P ] ratio at a high level, typically of the order of 500 M f For this ratio. 

ATP hydrolysis is non-linear with respect to time. This is not due to enzyme inactivation since progress curves, where the product of variable enzyme concentration and time is plotted against the amount of product, yield points that fall on a single line. This is a response expected if a competitive inhibition occurs during catalysis [80] and suggests an explanation for non-linearity since ADP is a competitive inhibitor with respect to ATP [19]. However, the presence of an ATP-generating system, which increases the rate of phosphate production by a factor of three, does not result in a linear rate [81]. 

ATP-generating system Creatine kinase (Boehringer Mannheim) 4mg/ml in glycerol/water (1 -i-l) store in aliquots of 200pi creatine phosphate (800 mM) 261.76 mg/ml in water, store in aliquots of 200 pi mix 1 part of CP and one part of CK. 

The biosynthesis of theanine from glutamate and ethylamine is catalyzed by theanine synthetase (L-glutamate ethylamine ligase, EC 6.3.1.6), which is an ATP-dependent enzyme and quite unstable. All attempts to biosynthesize theanine using this enzyme should overcome the problems of the ATP generating system. 

In an important experiments Nirenberg and Matthaei, in 1961, isolated ribosomes from E. coli and mixed them with crude extracts of soluble materials, also from E. coli cells. The extracts included tRNA molecules and aminocyl-tRNA synthases. The 20 amino acids, ATP, and an ATP-generating system (PEP + pyruvate kinase) were added. Nirenberg showed that under such conditions protein was synthesized by ribosomes in response to the presence of added RNA. For example, RNA from tobacco mosaic virus (Chapter... 

Nitrogenase catalyzes the hydrolysis of ATP ADP + P in a reaction that is reductant-dependent 15,29, 30) and results in electron transfer and activation to provide a low-potential species capable of reducing a unique range of substrates see Reducible Substrate section). The ratedetermining step of ATP utilization may be bimolecular (25, 31). The product ADP is an inhibitor of ATP utilization 32), and thus an ATP-generating system is used in vitro to reconvert ADP to ATP (15, 33). 

Figure 1. Synthesis of CoA from pantothenic acid and cysteine coupled with ATP-generating system...

Nitrogenase is a complex enzyme which catalyzes the reduction of a variety of substrates (Table IV) with the concomitant hydrolysis of ATP. It contains two, easily separable, iron-sulfur proteins one of which contains molybdenum. These require a reductant, a reducible substrate, an ATP-generating system, and an anaerobic environment to function. The ATP-generating system is necessary because ADP inhibits nitrogenase activity. Two important discoveries have helped to simplify the assay for nitrogenase. First, sodium dithionite proved to be an adequate reductant to replace the complex and unstable pyruvate phosphoroclastic system (Bulen et al., 1965) and, second, nitrogenase reduces acetylene to ethylene (Dilworth, 1966 Schollhorn and Burris, 1967) which can be measured at nanomolar concen-... 

Fig. 8. Azotobacter flavodoxin in the presence of Ac I, Ac2, and an ATP-generating system, uv-visible spectrum of (I) hydroquinone, (2) semiquinone, (3) oxidized (from Yates, 1972b).

Fig. 1 Stages of sea urchin sperm chromatin decondensation in vitro. (A-C) Demembranated sperm nuclei were incubated for various times in S oo containing an ATP-generating system and Hoechst 33342 to visualize the DNA. (A) Input condensed nucleus (B) partially decondensed nucleus (C) decondensed nucleus. (D) Swollen male pronucleus produced as described in Section VI,A. Scale bar, 10 m.

Demembranated sperm nuclei are decondensed for 1 hr in 20 ju.1 of Sioo as described in Section IV, then 2 fil of concentrated MVs is added with the ATP-generating system, and allowed to bind to chromatin for 40 min at room temperature. To separate chromatin-bound from unbound vesicles, the extract is underlaid with a equivalent volume of 1 M sucrose in TN buffer and nuclei centrifuged through the sucrose cushion at 500 g for 30 min at 4°C in a swinging-bucket rotor of an lEC CRU-5000 centrifuge (International Equipment Co). The pelleted chromatin is resuspended in egg lysis buffer and membranes labeled as described in Section V,A or directly viewed under fluorescence if prelabeled MVs are used. 

Note. In contrast to chromatin decondensation, vesicle binding to chromatin requires ATP but not ATP hydrolysis (Collas and Poccia, 1995a). Thus, the ATP-generating system may be replaced by 2 mM ATPyS or 2 mAf AMP-PNP (see Section IV,A,1 for references). Under these conditions, however, MV fusion will not occur on addition of GTP as described below. 

Fusion of chromatin-bound vesicles in vitro is promoted by GTP hydrolysis in sea urchin egg extracts (Collas and Poccia, 1995a), as shown previously in Xenopus (Boman et al., 19%). MVs are bound to chromatin in the presence of an ATP-generating system as described above. To induce fusion, GTT (Type II, Sigma G8752) is added to the extract from a frozen 5 mM stock in egg lysis buffer to a final concentration of 100 pM (Cameron and Poccia, 1994 Collas and Poccia, 1995a). Fusion usually occurs within 15-20 min of incubation at room temperature, but we routinely allow fusion to proceed for up to 40 min to ensure completion of the reaction. MV fusion can be assessed directly by... 

Sea Urchin. Sperm chromatin is incubated for 40 min in an Sio containing an ATP-generating system and 100 fiM GTP to promote decondensation, MV binding to chromatin, and fusion. If no further treatment is applied, these nuclei remain small ( 4 /i.m in diameter), do not contain a lamina, and are incapable... 

Pronuclear swelling and lamina assembly require ATP hydrolysis thus the ATP-generating system cannot be replaced by ATP analogs or GTP (Collas and Poccia, 1995b). 

Sperm nuclei are decondensed in 500 /il of an S o extract in a 1.5-ml centrifuge tube, and allowed to assemble a nuclear envelope in the presence of an ATP-generating system and GTP as described earlier. Nuclear swelling is promoted after 40 min as reported in Section VI,A. Swelling pronuclei are allowed to settle to the bottom of the tube for 1.5 hr. Nuclei free of membranes, or containing bound or fused membranes only, can be processed similarly. We let nuclei settle to the bottom of the tube, rather than centrifuge them, to minimize damage to the nuclear envelopes. 

Viruses multiply by synthesising their separate components which then have the capacity for self-assembly. On the other hand, cells multiply by binary fission. Viruses do not contain any ATP-generating system and they obtain their ATP from the cells they infect. 

A controversy has arisen about the participation of the mitochondria in thyroxine s stimulation of protein synthesis in cell-free systems. Whereas some investigators claimed to have observed the effect in microsomal preparations completely devoid of mitochondria, others claim that the presence of mitochondria in the cell-free preparation is indispensable to stimulation. The mitochondria do not act simply by generating ATP because their replacement by a classical ATP-generating system does not contribute to the stimulation of protein synthesis in microsomes of thyroidectomized animals injected with thyroxine. 

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