Biochemistry (Moscow) (v.74, #1)

Biochemical and molecular characterization of plant MYB transcription factor family by Hai Du; Li Zhang; Lei Liu; Xiao-Feng Tang; Wen-Jie Yang; Yan-Min Wu; Yu-Bi Huang; Yi-Xiong Tang (1-11).
MYB genes are widely distributed in higher plants and comprise one of the largest transcription factors, which are characterized by the presence of a highly conserved MYB domain at their N-termini. Over recent decades, biochemical and molecular characterizations of MYB have been extensively studied and reported to be involved in many physiological and biochemical processes. This review describes current knowledge of their structure characteristic, classification, multi-functionality, mechanism of combinational control, evolution, and function redundancy. It shows that the MYB transcription factors play a key role in plant development, such as secondary metabolism, hormone signal transduction, disease resistance, cell shape, organ development, etc. Furthermore, the expression of some members of the MYB family shows tissuespecificity.
Keywords: MYB transcription factors; classification; secondary metabolism; multi-functionality; signal transduction

IgA-specific proteins of pathogenic bacteria by T. N. Kazeeva; A. B. Shevelev (12-21).
Data on structure and specificity of bacterial IgA receptors (IgA-binding M-like proteins Arp4 and Sir22 from hemolytic streptococci of serogroup A, β-antigen from hemolytic streptococci of serogroup B, and SSL family proteins from Staphylococcus aureus) are surveyed in this review. The principal conclusion derived from comparison is the fact that all bacterial receptors bind the same site in the IgA molecule overlapping with the binding site of endogenous human IgA receptor CD89. We assume that this site, consisting of spatially close amino acid strands Leu257-Gly259 in domain Cα2 and Pro440-Phe443 in domain Cα3, is subject to conformational rearrangement induced by the binding of antigen in the IgA active site.
Keywords: IgA; IgA-binding proteins; CD89

Identification of a novel gene, MSAG, regulated by high levels of glucose and insulin by Xiao Yan Cui; Jin Xin Chen; Bing Wen Liu; Li Ying Xiao; Ding Zhi Fang (22-28).
Identification and characterization of novel genes involved in derangement of metabolisms of glucose and triglycerides are important in understanding the development of metabolic syndrome (MS) and atherosclerosis. Model rats with certain phenotypes of MS were fed a high-carbohydrate diet. The rat hepatic subtracted cDNA libraries were constructed and screened. A novel cDNA of full length was identified by screening of a human hepatic cDNA library with a mixture of probes of the differentially expressed fragments from the rat hepatic subtracted cDNA libraries. The corresponding gene of the cDNA was temporarily named metabolic syndrome-associated gene (MSAG). The predicted protein encoded by MSAG contains 110 amino acids and has a theoretical molecular weight of 11667.04 and an isoelectric point of 4.91. Compared with the housekeeping gene of β-actin, MSAG had low transcription activity. However, the mRNA level of MSAG in HepG2 cells, a human hepatoma cell line, was significantly increased by glucose and decreased by insulin concentrations higher than physiological levels. These results suggest that MSAG may be involved in the metabolism and/or its regulation of glucose, the functioning of insulin under non-physiological conditions, and further in the development of metabolic syndrome.
Keywords: differentially expressed genes; cDNA library; screening; glucose; metabolic syndrome-associated gene; HepG2 cells; real-time quantitative PCR

Insulin and insulin-like growth factors (IGFs) bind to their cognate receptors with high affinities, but due to their homology they may cross-react with each other’s receptors. We performed a series of binding studies to reanalyze the cross-reactivity of insulin, IGF-I, and IGF-II to affinity-purified insulin (IR) and type 2 IGF receptors (IGF-2R) from human placental membranes. IR and IGF-2R were purified using insulin- and mannose-6-phosphate affinity chromatography (I-AC and M6P-AC). Binding studies were performed with 125I-labeled and unlabeled ligands. According to immunoblotting, the only receptor species isolated by I-AC was IR, whereas the only receptor isolated by M6P-AC was IGF-2R. Isolated IR reacted to similar extent with 125I-labeled insulin and 125I-labeled IGF-II and significantly less with 125I-labeled IGF-I, implicating predominance of IR-A. The affinity of IR towards heterologous ligands increased after its separation from other membrane proteins. Affinity-purified IGF-2R was almost unable to bind ligands under experimental conditions used in this work, but when incubated with 125I-labeled ligands prior to affinity chromatography, IGF-2R interacted not only with IGF-II, but to a certain extent with the other two ligands. In the competitive M6P-AC, the binding of labeled ligands was inhibited with either homologous or heterologous ligands, in a dose dependent manner. In competitive ligand-blotting, specific interactions between 125I-labeled insulin and IR, and 125I-labeled IGF-II and IGF-2R were also inhibited with all unlabeled ligands, although to a different extent. The results presented in this work imply that isolation of IR an IGF-2R from their membrane milieu increases their reactivity towards all members of the insulin/IGF ligand family.
Keywords: insulin; insulin-like growth factors; receptors; affinity chromatography; ligand-binding studies

Enantioselective reductive amination of α-keto acids by papain-based semisynthetic enzyme by Chun-Xiang Chen; Bo Jiang; C. Branford-White; Li-Min Zhu (36-40).
Alkylation of a cysteine residue in papain with a pyridoxamine (PX) cofactor was carried out. The resulting semisynthetic enzyme (papain-PX) has no detectable protease activity but has the ability to catalyze enantioselective reductive amination of α-keto acids. The conjugate was characterized by ion-exchange chromatography, and the optimal reaction conditions were found. We report that papain-PX reductively aminates the alkyl side chain of functionalized α-keto acids to give the respective α-amino acids with high enantioselectivities, greater than 70%. Based on these studies, we propose a new model for the catalytic activity of the semisynthetic enzyme with Interchem software. The results of the study demonstrate the effectiveness of the modified enzyme and its potential for engineering new catalytic specificity.
Keywords: semisynthetic enzyme; papain; pyridoxamine; chemical modification

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation by M. A. Rosenfeld; V. B. Leonova; M. L. Konstantinova; S. D. Razumovskii (41-46).
The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.
Keywords: fibrinogen; fibrin; self-assembly; oxidation; structure

Induction of Arabidopsis gdh2 gene expression during changes in redox state of the mitochondrial respiratory chain by V. I. Tarasenko; E. Yu. Garnik; V. N. Shmakov; Yu. M. Konstantinov (47-53).
Expression of the gdh2 gene encoding the α-subunit of mitochondrial glutamate dehydrogenase depends on redox state of the mitochondrial electron transport chain. Treatment of Arabidopsis thaliana cell suspension with antimycin A, a respiratory chain complex III inhibitor, resulted in an increase in gdh2 transcripts within 2 h. Inhibition of complex I by rotenone did not influence the transcript level, but treatment with potassium cyanide, a complex IV inhibitor, also increased the transcript content. Thus, gdh2 gene expression obviously responds to changes in the respiratory chain segment localized between complexes I and III. Lack of activation of gene expression after treatment of a cell suspension with hydrogen per- oxide and the prooxidant paraquat and results of experiments with antioxidants suggest that gdh2 gene expression is not associated with increased content of reactive oxygen species generated during inhibition of the electron transport chain. Protein phosphorylation by serine/threonine protein kinases is the essential step required for signal transduction into nucleus resulting in the induction of gdh2 expression.
Keywords: glutamate dehydrogenase; alternative oxidase; respiratory chain; retrograde regulation; redox regulation; antimycin A

The structure of the intact heterotrimeric translation initiation factor 2 (e/aIF2) is of great interest due to its key role in the initiator tRNA delivery to the ribosome and in translation initiation regulation in eukaryotes and archaea. We have chosen aIF2 from the hyperthermophilic archaeobacterium Sulfolobus solfataricus (SsoIF2) as an object for crystallization and structural investigations. Genes of the SsoIF2 subunits α, β, and γ were cloned and superexpressed. A method for heterotrimer SsoIF2αβγ purification was elaborated with at least 95% purity. Highly ordered crystals of the full-sized SsoIF2, reflecting X-rays at the resolution up to 2.8 Å, were obtained for the first time.
Keywords: translation initiation; e/aIF2; heterotrimer; Sulfolobus solfataricus ; SsoIF2 crystals

In this study, we cloned the OsHAL3 gene from rice Oryza sativa. Alignment analysis revealed that OsHAL3 has a high sequence identity to Dfp protein in Escherichia coli and AtHAL3a protein in Arabidopsis thaliana, which have 4′-phosphopantothenoylcysteine decarboxylase (PPC-DC) activity. OsHAL3 can complement mutation in the E. coli dfp gene encoding PPC-DC, so that the mutant strains with OsHAL3 can grow on rich media at 42°C and on VB minimal media at 30°C. Complementation tests with point mutations of OsHAL3 suggested that the conserved Cys176 residue of OsHAL3 is a key active-site residue. The mutant OsHAL3 G180A has a partly reduced activity. Related mRNA-level analysis showed that the OsHAL3 gene is induced by calcium pantothenate in rice.
Keywords: OsHAL3 ; 4′-phosphopantothenoylcysteine decarboxylase; coenzyme A; rice

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: Structural properties and pigment-protein interactions by A. A. Zabelin; T. Y. Fufina; L. G. Vasilieva; V. A. Shkuropatova; M. G. Zvereva; A. Y. Shkuropatov; V. A. Shuvalov (68-74).
Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H A . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls PA and PB in the P+ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the PA and PB macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P+Q/PQ spectra suggests the 173-ester group of the bacteriochlorophyll PA to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll BA and the bacteriopheophytin HA. Structural changes are observed in the monomer bacteriochlorophyll BB binding site in the inactive chromophore branch of the mutant RCs.
Keywords: Rhodobacter sphaeroides ; mutant reaction centers; bacteriochlorophyll a ; trans-esterification reaction; covalent bond

High human GLUT1, GLUT2, and GLUT3 expression in Schizosaccharomyces pombe by Yuxin Yang; Zongli Hu; Zhizhao Liu; Yi Wang; Xuqing Chen; Guoping Chen (75-80).
In this study, three subfamily members of the human 12-transmembrane-domain cell-surface receptors GLUT1, GLUT2, and GLUT3 were heterologously expressed in the fission yeast Schizosaccharomyces pombe utilizing GST-GLUT fusion proteins. These fusion proteins were driven by the full-length nmt1 promoter (Pnmt1) derived from S. pombe. The transcription levels of the GST-GLUT fusion proteins were very high upon induction by removing thiamine from the media. One-step purification of the recombinant fusion proteins was achieved by GST-affinity chromatography. Approximately 300 µg of highly purified fusion protein were obtained from 3 g of wet cell paste (1 liter of cell culture), indicating that human membrane proteins can be efficiently expressed and purified in the fission yeast. With its available extensive genetic information and ease of genetic manipulation, the fission yeast is potentially a highly efficient host to express eukaryotic membrane proteins.
Keywords: GLUT; Schizosaccharomyces pombe ; membrane protein expression; GST-affinity chromatography

In this study, atrC (a novel gene from Azospirillum brasilense identified in our laboratory) was expressed in Escherichia coli, and SDS-PAGE analysis of the expressed AtrC revealed the apparent molecular weight of 45 kD. When analyzed under non-denaturing PAGE conditions and using L-tryptophan as a substrate, the purified AtrC protein exhibited aminotransferase activity, while crude protein extracts from A. brasilense Yu62 showed two activity bands with molecular masses estimated as 44 and 66 kD. Thus, we deduced that AtrC protein is identical to the 44 kD band of crude protein extracts. The optimal temperature and pH for the catalytic activity of the purified AtrC are 30°C and pH 7.0, respectively.
Keywords: function analysis; aminotransferase; Azospirillum brasilense ; indole-3-acetic acid

SsoII-like DNA-methyltransferase Ecl18kI: Interaction between regulatory and methylating functions by E. A. Fedotova; A. S. Protsenko; M. V. Zakharova; N. V. Lavrova; A. V. Alekseevsky; T. S. Oretskaya; A. S. Karyagina; A. S. Solonin; E. A. Kubareva (85-91).
The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Δ(72–379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.
Keywords: regulation in restriction-modification systems; (cytosine-5)-DNA methyltransferase; DNA-protein interactions

Replication protein A modulates the activity of human telomerase in vitro by M. P. Rubtsova; D. A. Skvortsov; I. O. Petruseva; O. I. Lavrik; P. V. Spirin; V. S. Prasolov; F. L. Kisseljov; O. A. Dontsova (92-96).
Our aim was to investigate how replication protein A (RPA) in a wide range of concentration can regulate the activity of human telomerase. We used an in vitro system based on human cell extracts with or without RPA. It has been shown that removal of RPA leads to loss of telomerase activity and addition of RPA restores telomerase activity and at the same time promotes telomerase processivity. However, high excess of RPA inhibited telomerase processivity and caused the synthesis of relatively short DNA fragments (about 50–100 nucleotides). We assume that, together with other telomere-binding proteins, RPA may take part in activation of telomere overhang elongation by telomerase at a certain stage of a cell cycle as well as in regulation of telomere length.
Keywords: telomerase; RPA; telomeres; regulation

Catalytic activity and stability of xanthine oxidase in aqueous-organic mixtures by M. R. Rashidi; M. H. Soruraddin; F. Taherzadeh; A. Jouyban (97-101).
In the present study, bovine milk xanthine oxidase activity in various aqueous-organic mixtures and the effects of pH, temperature, and lyophilization on the enzyme activity have been investigated. The enzyme was incubated with xan-thine as the substrate in Sorenson’s phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and the activity was determined spectrophotometrically in the absence and presence of different fractions of nine water-miscible organic solvents at 27–50°C and at different pH values ranging from 6 to 9. The organic solvents reduced the enzyme activity to different extents. In spite of these inhibitory effects, the enzyme showed relatively good stability in the aqueous-organic mixtures compared with the aqueous medium. A significant increase in the activity of the lyophilized enzyme was observed in pure organic solvents.
Keywords: xanthine oxidase; aqueous-organic mixtures; enzyme activity; thermal stability; lyophilization

Kunitz-type trypsin inhibitor with high stability from Spinacia oleracea L. seeds by Zhuang Kang; Jia-hong Jiang; Dong Wang; Ke Liu; Lin-fang Du (102-109).
The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K i = 2.3·10−6 M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.
Keywords: Spinacia oleracea L.; trypsin inhibitor; protein stability; Kunitz family; Pieris rapae

Glycobiology by G. Ya. Wiederschain (110-111).

DNA polymerase iota-like activity in crude cell extracts of different mouse organs by L. V. Gening; A. N. Petrochenkov; A. B. Reshetnyak; L. E. Andreeva; V. Z. Tarantul (114-114).

Erratum to: A false note of DNA polymerase iota in the choir of genome caretakers in mammals by L. V. Gening; A. V. Makarova; A. M. Malashenko; V. Z. Tarantul (115-115).