Adenylate Kinase

Adenylate kinase (AK) is involved in an essential step in nucleotide metabolism, catalysing the reversible transfer of the terminal phosphate group from ATP to AMP. The aim of this study was to describe 2-amino-4-methyl-N'-arylidene-thiazole-5-carbohydrazides effects on the activity of recombinant adenylate kinase from S. pneumoniae (AK SP) and E. coli (AK EC). Inhibition of AK SP and AK EC activity with synthetic compounds were performed against enzymes over-expressed in E. coli. The activity of recombinant enzymes was determined colorimetrically with 2,4-dinitrophenylhydrazine. 2-amino-4-methyl-N'-arylidene-thiazole-5-carbohydrazides inhibit AK SP and AK EC with different efficiencies. 2-amino-4-methyl 5-o-metoxi-phenyl-hydrazido thiazol (C 13 H 15 N 4 O 2 S) (compound I) binds tighter than 2-amino-4-methyl p-nitro-phenyl-hydrazido thiazol (C 12 H 11 N 5 O 3 S) (compound II). The I 50 values (the inhibitor concentration that leads to 50% activity inhibition) for AK SP was 34.8 µM for compound I and 178 µM for compound II. The I 50 AK EC was 38.63 µM for compound I and 170 µM for compound II. Rezumat Adenilat kinaza intervine într-o etapă importantă a metabolismului nucleotidelor purinice catalizând transferul reversibil al unei grupări fosfat terminale de pe ATP pe AMP. Obiectivul principal al acestui studiu a fost caracterizarea efectului a doi derivaţi 2-amino-4-metil-N'-ariliden-tiazolil-5-carbohidrazidici faţă de activitatea unor adenilat kinaze recombinante din S. pneumoniae (AK SP) şi E. coli (AK EC). Inhibarea activităţii AK SP şi AK EC a fost testată faţă de enzimele supra-exprimate în E. coli. Activitatea enzimelor recombinante a fost determinată colorimetric cu 2,4-dinitrofenilhidrazină. Derivaţii 2-amino-4-metil-5-carbonidrazol tiazol inhibă AK SP şi AK EC cu eficienţă diferită. 2-amino-4-metil-5 o-metoxi-fenil-hidrazido tiazol (C 13 H 15 N 4 O 2 S) (compus I) se leagă mai eficient decât 2-amino-4-metil p-nitro-fenil-hidrazido tiazol (C 12 H 11 N 5 O 3 S) (compus II). Valorile I 50 (concentraţia de inhibitor care determină inhibarea activităţii cu 50%) pentru AK SP a fost 34,80 µM pentru compusul I şi 178 µM pentru compusul II. The I 50 AK EC a fost 38,63 µM pentru compusul I şi 170 µM pentru compusul II.

Dysfunction of cardiac energy metabolism plays a critical role in many cardiac diseases, including heart failure, myocardial infarction and ischemia-reperfusion injury and organ transplantation. The characteristics of these diseases can be elucidated in vivo, though animal-free in vitro experiments, with primary adult or neonatal cardiomyocytes, the rat ventricular H9c2 cell line or the mouse atrial HL-1 cells, providing intriguing experimental alternatives. Currently, it is not clear how H9c2 and HL-1 cells mimic the responses of primary cardiomyocytes to hypoxia and oxidative stress. In the present study, we show that H9c2 cells are more similar to primary cardiomyocytes than HL-1 cells with regard to energy metabolism patterns, such as cellular ATP levels, bioenergetics, metabolism, function and morphology of mitochondria. In contrast to HL-1, H9c2 cells possess beta-tubulin II, a mitochondrial isoform of tubulin that plays an important role in mitochondrial function and regulati...

Diverse classes of proteins function through large-scale conformational changes and various sophisticated computational algorithms have been proposed to enhance sampling of these macromolecular transition paths. Because such paths are curves in a high-dimensional space, it has been difficult to quantitatively compare multiple paths, a necessary prerequisite to, for instance, assess the quality of different algorithms. We introduce a method named Path Similarity Analysis (PSA) that enables us to quantify the similarity between two arbitrary paths and extract the atomic-scale determinants responsible for their differences. PSA utilizes the full information available in 3N-dimensional configuration space trajectories by employing the Hausdorff or Fréchet metrics (adopted from computational geometry) to quantify the degree of similarity between piecewise-linear curves. It thus completely avoids relying on projections into low dimensional spaces, as used in traditional approaches. To elucidate the principles of PSA, we quantified the effect of path roughness induced by thermal fluctuations using a toy model system. Using, as an example, the closed-to-open transitions of the enzyme adenylate kinase (AdK) in its substrate-free form, we compared a range of protein transition path-generating algorithms. Molecular dynamics-based dynamic importance sampling (DIMS) MD and targeted MD (TMD) and the purely geometric FRODA (Framework Rigidity Optimized Dynamics Algorithm) were tested along with seven other methods publicly available on servers, including several based on the popular elastic network model (ENM). PSA with clustering revealed that paths produced by a given method are more similar to each other than to those from another method and, for instance, that the ENM-based methods produced relatively similar paths. PSA applied to ensembles of DIMS MD and FRODA trajectories of the conformational transition of diphtheria toxin, a particularly challenging example, showed that the geometry-based FRODA occasionally sampled the pathway space of force field-based DIMS MD. For the AdK transition, the new concept of a Hausdorff-pair map enabled us to extract the molecular structural determinants responsible for differences in pathways, namely a set of conserved salt bridges whose charge-charge interactions are fully modelled in DIMS MD but not in FRODA. PSA has the potential to enhance our understanding of transition path sampling methods, validate them, and to provide a new approach to analyzing conformational transitions.

Patients with chronic obstructive pulmonary disease (COPD), acute lung injury and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratio and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, while MuRF1-/- mice exposed to high CO2 did not develop muscle atrophy. AMPK, a metabolic sensor, was activated in myotubes exposed to high CO2 and loss-of-function studies showed that AMPK-α2 isoform is necessary for MuRF1 up-regulation and myofiber size reduction. High CO2 induced AMPK-α2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.

Some historical aspects of development of the concepts of functional coupling, metabolic channelling, compartmentation and energy transfer networks are reviewed. Different quantitative approaches, including kinetic and mathematical modeling of energy metabolism, intracellular energy transfer and metabolic regulation of energy production and fluxes in the cells in vivo are analyzed. As an example of the system with metabolic channelling, thermodynamic aspects of the functioning the mitochondrial creatine kinase functionally coupled to the oxidative phosphorylation are considered. The internal thermodynamics of the mitochondrial creatine kinase reaction is similar to that for other isoenzymes of creatine kinase, and the oxidative phosphorylation process specifically influences steps of association and dissociation of MgATP with the enzyme due to channelling of ATP from adenine nucleotide translocase. A new paradigm of muscle bioenergetics-the paradigm of energy transfer and feedback s...

Anthropological studies were done on 1276 Libyans from the Mediterranean cities of Tripoli and Benghazi, and from Sabha southward in The Sahara. The incidences of hemoglobin (Hb)-S and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency were low in the coastal areas and significantly high in Sabha. Hb-C occurred sporadically in Tripoli and Sabha, and was absent from Benghazi in the east. One case of Hb-J Benghazi was noted. There were no significant differences in the ABO blood group and Rh0 (D) type distributions in the three localities. G-6-PD gene GdA frequency was significantly high in Sabha. The lowest value of 6-phosphogluconate dehydrogenase (6-PGD) gene PGDA frequency and highest value of the gene PGDC were in Sabha. Adenylate kinase (AK) gene AK2 was only detectable in Tripoli. Acid phosphatase (AP) gene Pa frequency in Sabha was more than twice that in Tripoli and Benghazi, while Pc was distinctly lower in Sabha than in the northern cities. Haptoglobin gene Hp1 frequency was almost identical in all areas. Anthropometric measurements revealed overall homogeneity of the three samples, closer similarity in the coastal region to adjacent North African populations, and Negroid influence in the Saharan Libyans. Anthropometry substantiated findings from blood markers.