Adenosine Triphosphate Biosynthesis Research Articles

An ATP "Synthase" Derived from a Single Structural Domain of Bacterial Histidine Kinase.

ATP (adenosine triphosphate) is a vital energy source for living organisms, and its biosynthesis and precise concentration regulation often depend on macromolecular machinery composed of protein complexes or complicated multidomain proteins. We have identified a single-domain protein HK853CA derived from bacterial histidine kinases (HK) that can catalyze ATP synthesis efficiently. Here, we explored the reaction mechanism and multiple factors that influence this catalysis through a combination of experimental techniques and molecular simulations. Moreover, we optimized its enzymatic activity and applied it as an ATP replenishment machinery to other ATP-dependent systems. Our results broaden the understanding of ATP biosynthesis and show that the single CA domain can be applied as a new biomolecular catalyst used for ATP supply.

Molecular pharmacology of coenzyme Q10 in the context of treatment of hyperlipidemic conditions

Background. Therapy of hyperlipidemia with synthetic statin drugs is characterized by a number of side effects. In particular, statin-dependent myopathy occurs as a result of statin inhibition of biosynthesis of cofactor coenzyme Q10 (CoQ10), which plays a central role in the electron transport chains during adenosine triphosphate (ATP) synthesis in mitochondria.Objective: systematic analysis of scientific publications on CoQ10 pharmacology with an emphasis on its effect on the lipid profile.Material and methods. The analysis included 16,788 articles found by “coenzyme Q10 OR ubiquinone" query in the PubMed/MEDLINE biomedical publications database. Topological and metric big data analysis methods were used developed in the scientific school of Academician of the Russian Academy of Sciences Yu.I. Zhuravlev.Results. A much wider range of CoQ10 pharmacological effects was established than simply supporting ATP biosynthesis. Coenzyme Q10 exhibits pronounced lipid-lowering, anti-asthenic, and anti-inflammatory effects and can be successfully used in the treatment of asthenic conditions, cardiovascular pathology (including hyperlipidemia and statin-induced myopathy), carbohydrate metabolism disorders (insulin resistance), and diverse neurological diseases. CoQ10 supplements reduce mortality by 50% in patients with cardiovascular diseases, improve glycemic control in insulin resistance, kidney function, and are useful in the treatment of migraine and neurodegenerative pathologies (Parkinson's disease, etc.).Conclusion. Experimental data and evidence base indicate the potential of using CoQ10 in patients with hyperlipidemia and other cardiovascular system diseases.

Electroacupuncture-modulated extracellular ATP levels in prefrontal cortex ameliorated depressive-like behavior of maternal separation rats

Maternal separation (MS) is a type of early-life stress that has been linked to neuropsychiatric disorders, especially depression. Increasing evidence indicates that the adenosine triphosphate (ATP) level in the prefrontal cortex (PFC) is involved in the pathophysiology of depression. To investigate the potential relationship between ATP in PFC and antidepressant effects of electroacupuncture (EA) treatment, we assessed genes involved in ATP biosynthesis as well as the extracellular ATP levels in a rat model exposed to neonatal MS. Our results demonstrated that reduced expression of ABCG2 (an ATP-binding cassette protein) and ATP levels in the PFC of depressive-like rats exposed to MS can be attenuated by EA stimulus at the Baihui (GV20) and Yintang (GV29) acupoints. Moreover, the antidepressant effect of EA treatment was blocked by administration of suramin, a broad purinergic P2 receptor antagonist. Together, these results suggested that electroacupuncture may be able to modulate extracellular ATP levels in the PFC of depressive-like MS rats, potentially contributing to its antidepressant effects.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15–33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g−1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L−1L-met and 0.60 g L−1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L−1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L−1 to 10.1 g L−1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

The photosynthesis apparatus of European mistletoe (Viscum album).

European mistletoe (Viscum album) is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (Complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). Here, we present an investigation of the V. album energy metabolism taking place in chloroplasts. Thylakoids were purified from young V. album leaves, and membrane-bound protein complexes were characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics. We identified >1,800 distinct proteins (accessible at https://complexomemap.de/va_leaves), including nearly 100 proteins forming part of the protein complexes involved in the light-dependent part of photosynthesis. The photosynthesis apparatus of V. album has distinct features: (1) comparatively low amounts of Photosystem I; (2) absence of the NDH complex (the chloroplast pendant of mitochondrial Complex I involved in cyclic electron transport (CET) around Photosystem I); (3) reduced levels of the proton gradient regulation 5 (PGR5) and proton gradient regulation 5-like 1 (PGRL1) proteins, which offer an alternative route for CET around Photosystem I; (4) comparable amounts of Photosystem II and the chloroplast ATP synthase complex to other seed plants. Our data suggest a restricted capacity for chloroplast ATP biosynthesis by the photophosphorylation process. This is in addition to the limited ATP supply by the mitochondria. We propose a view on mistletoe's mode of life, according to which its metabolism relies to a greater extent on energy-rich compounds provided by the host trees.

In vitro biosynthesis of ATP from adenosine and polyphosphate

Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively.Graphical

Melatonin elevated Sclerotinia sclerotiorum resistance via modulation of ATP and glucosinolate biosynthesis in Brassica rapa ssp. pekinensis

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SignificanceMelatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

Physical Activity-Dependent Regulation of Parathyroid Hormone and Calcium-Phosphorous Metabolism.

Exercise perturbs homeostasis, alters the levels of circulating mediators and hormones, and increases the demand by skeletal muscles and other vital organs for energy substrates. Exercise also affects bone and mineral metabolism, particularly calcium and phosphate, both of which are essential for muscle contraction, neuromuscular signaling, biosynthesis of adenosine triphosphate (ATP), and other energy substrates. Parathyroid hormone (PTH) is involved in the regulation of calcium and phosphate homeostasis. Understanding the effects of exercise on PTH secretion is fundamental for appreciating how the body adapts to exercise. Altered PTH metabolism underlies hyperparathyroidism and hypoparathyroidism, the complications of which affect the organs involved in calcium and phosphorous metabolism (bone and kidney) and other body systems as well. Exercise affects PTH expression and secretion by altering the circulating levels of calcium and phosphate. In turn, PTH responds directly to exercise and exercise-induced myokines. Here, we review the main concepts of the regulation of PTH expression and secretion under physiological conditions, in acute and chronic exercise, and in relation to PTH-related disorders.

Aurora kinase inhibitor restrains STAT5-activated leukemic cell proliferation by inducing mitochondrial impairment.

Current chemotherapy regimens on acute myeloid leukemia (AML) still have some drawbacks, such as intolerance and drug resistance, which calls need for the development of targeted therapy. Signal transducer and activator of transcription 5 (STAT5) is often overexpressed or abnormally activated in leukemia and involved in cell self-renewal, proliferation, and stress adaptation. Overexpressed Aurora A (AURKA) is associated with poor prognosis in tumors, and inhibitors against AURKA are already in clinical trials. However, it has rarely been reported whether AURKA inhibitors restrain STAT5-activated leukemia cells. In this study, we constructed STAT5 constitutively activated (cS5) cells and found that STAT5 promoted cell proliferation and colony formation. Moreover, cS5 cells showed elevated reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, which indicated higher mitochondrial metabolism in cS5 cells. A novel AURKA inhibitor AKI604 was synthesized and showed significant inhibitory effects to the proliferation and colony formation in both STAT5 constitutively activated and nonactivated AML cells. AKI604 induced mitochondrial impairment, leading to the disruption of mitochondrial membrane potential and the elevation of ROS as well as cellular calcium (Ca2+ ) levels. AKI604 could also decline basal oxygen consumption rate and ATP biosynthesis, indicating the damage of oxidative phosphorylation. Furthermore, AKI604 exhibited significant antitumor effect in the HL-60 cS5 xenograft model of the BALB/c nude mice without an obvious influence on mice body weight and other healthy indicators. This study suggested that AKI604 was a potential strategy to overcome STAT5-induced leukemic proliferation in AML treatment by inducing mitochondrial impairment.

Molecular Assembly of Rotary and Linear Motor Proteins.

Molecular machines are an important and emerging frontier in research encompassing interdisciplinary subjects of chemistry, physics, biology, and nanotechnology. Although there has been major interest in creating synthetic molecular machines, research on natural molecular machines is also crucial. Biomolecular motors are natural molecular machines existing in nearly every living systems. They play a vital role in almost every essential process ranging from intracellular transport to cell division, muscle contraction and the biosynthesis of ATP that fuels life processes. The construction of biomolecular motor-based biomimetic systems can help not only to deeply understand the mechanisms of motor proteins in the biological process but also to push forward the development of bionics and biomolecular motor-based devices or nanomachines. From combination of natural biomolecular motors with supramolecular chemistry, great opportunities could emerge toward the development of intelligent molecular machines and biodevices. In this Account, we describe our efforts to design and reconstitute biomolecular motor-based active biomimetic systems, in particular, the combination of motor proteins with layer-by-layer (LbL) assembled cellular structures. They are divided into two parts: (i) reconstitution of rotary molecular motor FOF1-ATPase, which is coated on the surface of LbL assembled microcapsules or multilayers and synthesizes adenosine triphosphate (ATP) through creating a proton gradient; (ii) molecular assembly of linear molecular motors, the kinesin-based active biomimetic systems, which are coated on a planar surface or LbL assembled tubular structure and drive the movement of microtubules. LbL assembled structures offer motor proteins with an environment that resembles the natural cell. This enables high activity and optimized function of the motor proteins. The assembled biomolecular motors can mimic their functionalities from the natural system. In addition, LbL assembly provides facile integration of functional components into motor protein-based active biomimetic systems and achieves the manipulation of FOF1-ATPase and kinesin. For FOF1-ATPase, the light-driven proton gradient and controlled ATP synthesis are highlighted. For kinesin, the strategies used for the direction and velocity control of kinesin-based molecular shuttles are discussed. We hope this research can inspire new ideas and propel the actual applications of biomolecular motor-based devices in the future.

Discovery and development of SCM-198, a novel compound from Chinese herb for ischemic stroke

Stroke especially ischemic stroke is a key factor for causing high mortality in Asian populations. Many therapeutic approaches have been investigated to solve this problem though it remains a main issue for treating stroke. Recently, we found a unique single compound Leonurine (named also as SCM-198, J CVP 2009, Atherosclerosis 2012a and Atherosclerosis 2012b) extracted from Chinese herb-herba leonuri could prevent (Stroke 2010) and treat ischemic stroke (to be submitted). Middle cerebral artery occlusion (MCAO) was used and the animals were pretreated with orally for 7 days and the surgery was done. SCM-198 pretreatment reduced infarct volume, improved neurological deficit in stroke groups, increased activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase, and decreased levels from the lipid peroxidation marker malondialdehyde. SCM-198 further inhibited mitochondrial reactive oxygen species production and adenosine triphosphate biosynthesis. In the acute treatment group, rats were treated by i.v. with SCM-198 15 mg/kg at 0.5h, 1h and 2h after surgery and continued for 7 days. PET/CT and the neurological deficit score showed a significant amelioration in the treated group compared to vehicle group. TTC staining after 3 days' treatment showed a reduced infarct volume. Results in immunohistochemical staining, Iba-1 immunoreactivity in hippocampal CA1 region were markedly decreased in the treated groups. Further, a significant better maintenance of neuronal structure in hippocampal CA1 region and fewer activated microglia in the treated groups (especially in the 0.5h group) was observed than the vehicle group. SCM-198 could go through BBB rapidly with T1/2 of 25.4mins analyzed by microdialysis. Our results demonstrated that SCM-198 not only served as a prophylactic agent for ischemic stroke but also a potential drug candidate for acute treatment of stroke.

Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury.

Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours. MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored.

Inosine Triphosphate Protects Against Ribavirin-Induced Adenosine Triphosphate Loss by Adenylosuccinate Synthase Function

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown. We examined whether ITP can be used by adenosine triphosphatase (ATPase) in human erythrocytes or recombinant human adenylosuccinate synthase (ADSS). RBV-induced adenosine triphosphate (ATP) reduction in erythrocytes was compared with the genetically determined low or normal activity of ITPA, leading respectively to high or normal ITP levels. Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR). Interestingly, the alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by the ADSS inhibitor 6-mercaptoethanol (6-MP). ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

Leonurine Protects Middle Cerebral Artery Occluded Rats Through Antioxidant Effect and Regulation of Mitochondrial Function

Oxidative stress is known to be involved in ischemic stroke. Intense interest is drawn to the therapeutic potential of Chinese herbs on ischemic stroke because many of them contain antioxidant properties. Leonurine, 1 of the active compounds from purified Herba Leonuri, was studied to evaluate its possible therapeutic effects on ischemic stroke. Method-Middle cerebral artery occlusion was selected as our model of study. The animals were pretreated with Leonurine orally for 7 days and the surgery was done. One day after surgery, 2,3,5-triphenyltetrazolium chloride staining and neurological deficit score were carried out to evaluate the functional outcome of animals, whereas levels of superoxide dismutase, glutathione peroxidase, and malondialdehyde were analyzed for oxidative stress analysis. For mitochondrial studies, 3 hours after surgery, mitochondria were isolated for analysis of reactive oxygen species production, adenosine triphosphate biosynthesis, oxygen consumption, and respiratory control ratio value. Result-In in vivo experiments, Leonurine pretreatment reduced infarct volume, improved neurological deficit in stroke groups, increased activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase, and decreased levels from the lipid peroxidation marker malondialdehyde. In terms of mitochondrial modulation, Leonurine inhibited mitochondrial reactive oxygen species production and adenosine triphosphate biosynthesis. Animal studies also demonstrated that the mitochondrial function and redox state were restored by Leonurine treatment. Leonurine has neuroprotective effects and carries a therapeutic potential of stroke prevention.

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion-cytochrome c-caspase protease mechanism.

Incorporating recipient choice in kidney transplantation.

Despite the acute shortage of cadaveric organs for kidney transplantation, more than 10% of cadaveric kidneys are discarded each year because of marginal quality. Transplant recipients' access to these kidneys and to information about their quality is limited. A Monte Carlo model was developed to simulate the operations of an organ procurement organization over a 10-yr period. Donor and recipient characteristics were generated from the United States Renal Data System. Kidneys were assigned one of five possible grades, which were determined by calculating the relative risk of graft failure associated with donor characteristics and HLA matching for every donor-candidate pair. Modeled were recipient decisions to accept or reject a kidney on the basis of the relative change in quality-adjusted life years (QALY). Compared were the United Network of Organ Sharing (UNOS) policy, the UNOS expanded donor criteria policy, two benchmark policies (one equity driven and the other efficiency driven), and a hybrid policy that incorporated recipient choice into the UNOS algorithm. Sensitivity analyses for major input variables were performed. Compared with UNOS, an algorithm that incorporated recipient choice predicted a 6% increase in QALY, a 12% decrease in median waiting time, a 39% increase in the likelihood of transplantation, and a 56% reduction in the number of discarded kidneys. Benefits were observed across categories of age, gender, and race. Incorporating recipient choice in kidney transplantation would improve equity, efficiency, and QALY of the end-stage renal disease population.

Experimental porphyria induced in rats by ethionine.

OUR earlier findings have shown that administration of adenine nucleosides or nucleotides to rats with different experimental porphyrias was followed by a decrease in excess porphyrin formation1–4. These observations have led us to the conclusion that these purine derivatives and particularly ATP play an important part in the quantitative regulation of porphyrin biosynthesis1. Moreover, it occurred to us that if this hypothesis is correct, administration of compounds capable of causing a decrease in the biosynthesis of adenosine triphosphate (ATP) might well bring about the formation of excess porphyrins and porphyrin precursors. Our observations with 6-mercapto-purine3 and orotic acid4 have confirmed this working hypothesis.

The Biosynthesis of Adenosine Triphosphate and Guanosine Triphosphate in the Rabbit Erythrocyte in Vivo and in Vitro

The Biosynthesis of Adenosine Triphosphate and Guanosine Triphosphate in the Rabbit Erythrocyte in Vivo and in Vitro