Biochemistry (Moscow) (v.80, #1)

Precise regulation is fundamental to the proper functioning of enzymes in a cell. Current opinions about this, such as allosteric regulation and dynamic contribution to enzyme regulation, are experimental models and substantially empirical. Here we proposed a theoretical and thermodynamic model of enzyme regulation. The main idea is that enzyme regulation is processed via the regulation of abundance of active conformation in the reaction buffer. The theoretical foundation, experimental evidence, and experimental criteria to test our model are discussed and reviewed. We conclude that basic principles of enzyme regulation are laws of protein thermodynamics and it can be analyzed using the concept of distribution curve of active conformations of enzymes.
Keywords: enzyme regulation; thermodynamics; active conformation; distribution curve

While some representatives of the animal kingdom were improving their biological mechanisms and properties for adapting to ever-changing life conditions, the genus Homo was developing backward: human individuals were losing their adaptation to life areas conquered earlier. Losing step-by-step their useful traits including the body hair cover, the primitive genus Homo retained his viability only under very favorable conditions of the equatorial Africa. Protection from UV radiation danger was provided only by pigmentation of skin, hair, and eyes. However, “impoverished” individuals of this genus gained the ability to walk upright. Their hands became free from participation in movement and became fine tools for producing useful instruments, from the stone knife to the computer. The major consequence of upright movement and hand development became the powerful development of the brain. A modern human, Homo sapiens, appeared capable of conquering very diverse new habitats. The human’s expansion on the Earth occurred somewhat limited by his dependence on vitamin D. His expansion into new areas with lower Sun activity was partially associated with the loss of skin pigmentation. But there is an open question, whether under these new conditions he is satisfactorily provided with vitamin D. This paper discusses the following problems: how can we ensure a sufficient intake of vitamin D, how much does an individual require for his existence and optimal life, what will be consequences of vitamin D deficiency, and what are the prospects for better provision with vitamin D?
Keywords: vitamin D (deficiency); vitamin D (supply); cholecalciferol; ergocalciferol; UV radiation

A long-lived metastable minor fraction has been detected and characterized in myeloma protein IgG4 MAM by hydro- and thermodynamic methods. The sedimentation constants of the minor and the major protein fractions are different. The stability of the two CH2 domains in the minor fraction varies. The unique characteristics of these IgG4 MAM conformers arise from the fact that on exchange of the heavy chains between IgG4 molecules, in some of them only one non-canonical bond Cys226-Cys229 is formed in the central part of the “hinge region” instead of two canonical interchain disulfide bonds Cys226-Cys226 and Cys229-Cys229. This leads to asymmetric structure of the IgG4 MAM molecules.
Keywords: immunoglobulin IgG4; CH2-domain; stability; carbohydrate moiety; disulfide bond

The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp→Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34–95% of the wild-type level) showed activity high enough (35–108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5–M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.
Keywords: yeast; plasma membrane; secretory vesicles; biogenesis; Pma1 H+-ATPase; H+ transport; site-directed mutagenesis

The objective of the present work was to determine whether it is possible to use a nonhydrolyzable analog of ATP (AMP-PNP) as an inhibitor of ATP-dependent scanning of the leader sequence of eukaryotic mRNA in translation initiation. The formation of ribosomal 48S initiation complexes at the start codon of the capped mRNA leader sequence of rabbit β-globin mRNA was studied. The study was carried out in a system composed of individual components of translation initiation. The dependences of the efficiency of formation of 48S initiation complexes on ATP concentration and incubation time were obtained in the absence and presence of AMP-PNP. It was found that AMP-PNP did not affect the efficiency of formation of 48S initiation complexes in all cases under study. We conclude that the uncleavable analog of ATP, AMP-PNP, is not an inhibitor of translation initiation in eukaryotes.
Keywords: translation initiation; 48S ribosomal initiation complex; AMP-PNP; toeprinting

To better understand how photosystem (PS) activity is regulated during state transitions in cyanobacteria, we studied photosynthetic parameters of photosystem II (PSII) and photosystem I (PSI) in Synechocystis PCC 6803 wild type (WT) and its mutants deficient in oxidases (Ox) or succinate dehydrogenase (SDH). Dark-adapted Ox mutant, lacking the oxidation agents, is expected to have a reduced PQ pool, while in SDH mutant the PQ pool after dark adaptation will be more oxidized due to partial inhibition of the respiratory chain electron carriers. In this work, we tested the hypothesis that control of balance between linear and cyclic electron transport by the redox state of the PQ pool will affect PSII photosynthetic activity during state transition. We found that the PQ pool was reduced in Ox mutant, but oxidized in SDH mutant after prolonged dark adaptation, indicating different states of the photosynthetic apparatus in these mutants. Analysis of variable fluorescence and 77K fluorescence spectra revealed that the WT and SDH mutant were in State 1 after dark adaptation, while the Ox mutant was in State 2. State 2 was characterized by ∼1.5 time lower photochemical activity of PSII, as well as high rate of P700 reduction and the low level of P700 oxidation, indicating high activity of cyclic electron transfer around PSI. Illumination with continuous light 1 (440 nm) along with flashes of light 2 (620 nm) allowed oxidation of the PQ pool in the Ox mutant, thus promoting it to State 1, but it did not affect PSII activity in dark adapted WT and SDH mutant. State 1 in the Ox mutant was characterized by high variable fluorescence and P700+ levels typical for WT and the SDH mutant, indicating acceleration of linear electron transport. Thus, we show that PSII of cyanobacteria has a higher photosynthetic activity in State 1, while it is partially inactivated in State 2. This process is controlled by the redox state of PQ in cyanobacteria through enhancement/inhibition of electron transport on the acceptor side of PSII.
Keywords: cyanobacteria; mutants; photosystem II; NADP+ ; state transitions; plastoquinone

Effect of trehalose on oxygen evolution and electron transfer in photosystem 2 complexes by M. D. Mamedov; I. O. Petrova; D. V. Yanykin; A. A. Zaspa; A. Yu. Semenov (61-66).
The pigment-protein complex of photosystem 2 (PS 2) catalyzes the light-driven oxidation of water molecule and the reduction of plastoquinone. In this work, we studied the effect of the disaccharide trehalose, which is unique in its physicochemical properties, on isolated PS 2 complex. It was found that trehalose significantly stimulated the steady-state rate of oxygen evolution. The study of single flash-induced fluorescence decay kinetics demonstrated that trehalose did not affect the rate of Q A oxidation, although it led to an increase in the relative fractions of PS 2 reaction centers capable of Q A oxidation. Trehalose also prevented PS 2 complexes from being inactivated on prolonged storage. We propose that in the presence of trehalose, which affects the extent of hydration, the protein can preferentially exist in a more optimal conformation for effective functioning.
Keywords: photosystem 2; water-oxidizing complex; oxygen evolution; trehalose; chlorophyll fluorescence; plastoquinone oxidation

Chimerical proteins consisting of enhanced yellow fluorescent protein (EYFP) connected by linkers of different length and nature to the N-terminal end of small heat shock protein HspB1 were obtained and characterized. To obtain fluorescent chimeras with properties similar to those of unmodified small heat shock protein, we used either 12-residue-long linkers of different nature (highly flexible Gly-Ser linker (L1), rigid α-helical linker (L2), or rigid Pro-Ala linker (L3)) or highly flexible Gly-Ser linker consisting of 12, 18, or 21 residues. The wild-type HspB1 formed large stable oligomers consisting of more than 20 subunits. Independent of the length or the nature of the linker, all the fluorescent chimeras formed small (5–9 subunits) oligomers tending to dissociate at low protein concentration. Chaperone-like activity of the wild-type HspB1 and its fluorescent chimeras were compared using lysozyme as a model protein substrate. Under the conditions used, all the fluorescent chimeras possessed higher chaperone-like activity than the wild-type HspB1. Chaperone-like activity of fluorescent chimeras with L1 and L3 linkers was less different from that of the wild-type HspB1 compare to the chaperone-like activity of chimeras with rigid L2 linker. Increase in the length of L1 linker from 12 up to 21 residues leads to decrease in the difference in the chaperone-like activity between the wild-type protein and its fluorescent chimeras. Since the N-terminal domain of small heat shock proteins participates in formation of large oligomers, any way of attachment of fluorescent protein to the N-terminal end of HspB1 leads to dramatic changes in its oligomeric structure. Long flexible linkers should be used to obtain fluorescent chimeras with chaperone-like properties similar to those of the wild-type HspB1.
Keywords: small heat shock proteins; HspB1; fluorescent chimeras; quaternary structure; chaperone-like activity

Quantitative analysis of structure-activity relationships of tetrahydro-2H-isoindole cyclooxygenase-2 inhibitors by V. R. Khayrullina; A. Ya. Gerchikov; A. A. Lagunin; F. S. Zarudii (74-86).
Using the GUSAR program, structure-activity relationships on inhibition of cyclooxygenase-2 (COX-2) catalytic activity were quantitatively analyzed for twenty-six derivatives of 4,5,6,7-tetrahydro-2H-isoindole, 2,3-dihydro-1H-pyrrolyzine, and benzothiophene in the concentration range of 0.6–700 nmol/liter IC50 values. Six statistically significant consensus QSAR models for prediction of IC50 values were designed based on MNA- and QNA-descriptors and their combinations. These models demonstrated high accuracy in the prediction of IC50 values for structures of both training and test sets. Structural fragments of the COX-2 inhibitors capable of strengthening or weakening the desired property were determined using the same program. This information can be taken into consideration on molecular design of new COX-2 inhibitors. It was shown that in most cases, the influence of structural fragments on the inhibitory activity of the studied compounds revealed with the GUSAR program coincided with the results of expert evaluation of their effects based on known experimental data, and this can be used for optimization of structures to change the value of their biological activity.
Keywords: cyclooxygenase-2 inhibitors; GUSAR; QSAR models; QNA and MNA descriptors; analysis of structure-activity relationships

Effect of insulin, the glutathione system, and superoxide anion radical in modulation of lipolysis in adipocytes of rats with experimental diabetes by V. V. Ivanov; E. V. Shakhristova; E. A. Stepovaya; O. L. Nosareva; T. S. Fedorova; N. V. Ryazantseva; V. V. Novitsky (87-96).
Spontaneous lipolysis was found to be increased in adipocytes of rats with alloxan-induced diabetes. In addition, isoproterenol-stimulated hydrolysis of triacylglycerols was inhibited against the background of oxidative stress and decreased redox-status of cells. A decrease in the ability of insulin to inhibit isoproterenol-stimulated lipolysis in adipocytes that were isolated from adipose tissue of rats with experimental diabetes was found, which shows a disorder in regulation of lipolysis in adipocytes by the hormone in alloxan-induced diabetes. Based on these findings, we concluded that there is an influence of reactive oxygen species, superoxide anion radical in particular, and redox potential of the glutathione system on molecular mechanisms of change in lipolysis intensity in rat adipocytes in alloxan-induced oxidative stress. Activation of spontaneous lipolysis under conditions of oxidative stress might be a reason for the high concentration of free fatty acids in blood plasma in experimental diabetes, and this may play a significant role in development of insulin resistance and appearance of complications of diabetes
Keywords: diabetes mellitus; adipocyte; oxidative stress; lipolysis; glutathione system; Mn-tetra(N-methyl-4-pyridyl)porphyrin; N-ethylmaleimide

Implication of α2β1 integrin in anoikis of MCF-7 human breast carcinoma cells by G. E. Morozevich; N. I. Kozlova; O. Y. Susova; P. A. Karalkin; A. E. Berman (97-103).
Silencing of α2β1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2β1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2β1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2β1 to the level of control cells. The data show for the first time that integrin α2β1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.
Keywords: integrins; anoikis; tumor growth; signal protein kinases

Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in “old” hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of “old” cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of agedependent insulin resistance.
Keywords: hepatocytes; insulin resistance; aging; myriocin; fumonisin B1; imipramine; GW4869

Conjugation with the new branched copolymers, PEG-chitosan and glycol-chitosan, is suggested to improve the therapeutic properties of L-asparaginase from Erwinia carotovora (EwA). The structure and composition of such conjugates were optimized for maximal catalytic efficiency (k cat/K M) under physiological conditions, yielding improvement by a factor of 3–6 compared to the native enzyme. This effect is attributed mainly to the shift of pH activity profile towards lower pH values due to the polycationic nature of the copolymer. The thermostability of EwA conjugates was also considerably improved. Chito-PEGylation, similarly to PEGylation, can be expected to improve pharmacokinetic properties and to reduce immunogenicity of this medically relevant enzyme. It is worth mentioning that a new versatile approach based on IR spectroscopy has been developed to determine PEG-chitosan copolymer composition as well as composition of copolymer-enzyme conjugates. The proposed analytic method is “reagent-free” and allows fast and reliable determination of parameters of interest from the single IR spectrum in contrast to laborious and unreliable methods based on polymer free amino group titration with TNBS and OPA.
Keywords: recombinant L-asparaginase; branched copolymers; PEG-chitosan; activity; thermostability; FTIR; HPLC; pH optima

The A2B adenosine receptor colocalizes with adenosine deaminase in resting parietal cells from gastric mucosa by R. M. Arin; A. I. Vallejo; Y. Rueda; O. Fresnedo; B. Ochoa (120-125).
The A2B adenosine receptor (A2BR) mediates biological responses to extracellular adenosine in a wide variety of cell types. Adenosine deaminase (ADA) can degrade adenosine and bind extracellularly to adenosine receptors. Adenosine modulates chloride secretion in gastric glands and gastric mucosa parietal cells. A close functional link between surface A2BR and ADA has been found on cells of the immune system, but whether this occurs in the gastrointestinal tract is unknown. The goal of this study was to determine whether A2BR and ADA are coexpressed at the plasma membrane of the acid-secreting gastric mucosa parietal cells. We used isolated gastric parietal cells after purification by centrifugal elutriation. The membrane fraction was obtained by sucrose gradient centrifugation. A2BR mRNA expression was analyzed by RT-PCR. The surface expression of A2BR and ADA proteins was evaluated by Western blotting, flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are expressed in cell membranes isolated from gastric parietal cells. They show a high degree of colocalization that is particularly evident in the surface of contact between parietal cells. The confocal microscopy data together with flow cytometry analysis suggest a tight association between A2BR and ADA that might be specifically linked to glandular secretory function.
Keywords: adenosine receptor; adenosine deaminase; purine nucleoside signaling; cell membrane; gastric mucosa parietal cell