Biochemistry (Moscow) (v.75, #7)
Lipid signaling in the atherogenesis context by A. N. Smirnov (793-810).
Normally macrophages localized in the arterial vessel wall perform the “reverse transfer” of cholesterol, which includes endocytosis of low density lipoproteins (LDL), cholesterol transfer to newly formed high density lipoprotein particles, and their following elimination by the liver. The homeostatic function of macrophages for cholesterol involves a system of lipid sensors. Oxysterol sensors LXRs, oxysterol and cholesterol sensors INSIG and SCAP acting through controlled transcription factors SREBP, as well as sensors for oxidized fatty acids and their derivatives, PPAR, are the best studied. Activation of LXR and PPAR is also accompanied by inhibition of macrophage functions related to inflammation. Accumulation of oxidized and otherwise modified LDL in the subendothelial space induced by endothelium injury, infection, or other pathogenic factors instead of stimulation of the homeostatic functions of macrophages leads to their weakening with a concurrent increase in the inflammatory potential of these cells. These shifts seem to drive the transformation of macrophages into foam cells, which form the core of sclerotic plaques. The intervention of another lipid sensor, TLR4, can trigger such a radical change in the functional activity of macrophages. The interaction of modified LDL with this signaling receptor results in inhibition of the homeostatic oxysterol signaling, induction of additional LDL transporters, and activation of the phagocytic function of macrophages. The re-establishment of cholesterol homeostasis under these circumstances can be achieved by administration of LXR and PPARγ agonists. Therefore, it is urgent to design ligands with reduced side effects.
Keywords: atherogenesis; macrophages; cholesterol; lipid sensors; oxysterols; modified low density lipoproteins
“Smart” liposomal nanocontainers in biology and medicine by Y. S. Tarahovsky (811-824).
The perspectives of using liposomes for delivery of drugs to desired parts of the human body have been intensively investigated for more than 30 years. During this time many inventions have been suggested and different kinds of liposomal devices developed, and a number of them have reached the stages of preclinical or clinical trials. The latest techniques can be used to develop biocompatible nano-sized liposomal containers having some abilities of artificial intellect, such as the presence of sensory and responsive units. However, only a few have been clinically approved. Further improvements in this area depend on our knowledge of the interactions of drugs with the lipid bilayer of liposomes. Further studies on liposomal transport through the human body, their targeting of cells requiring therapeutic treatment, and finally, the development of techniques for controlled drug delivery to desired acceptors on cell surfaces or in cytoplasm are still required.
Keywords: liposomes; intellectual nanocontainers; drugs targeting; nanomedicine
Inorganic polyphosphates in mitochondria by T. V. Kulakovskaya; L. P. Lichko; V. M. Vagabov; I. S. Kulaev (825-831).
Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (∼15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7–10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca2+/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting Pi from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.
Keywords: inorganic polyphosphate; mitochondria; polyP/Ca2+/polyhydroxybutyrate; exopolyphosphatase; membrane potential; glucose repression; yeast; animal; insect
Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of Rhodobacter sphaeroides by A. G. Yakovlev; L. G. Vasilieva; T. I. Khmelnitskaya; V. A. Shkuropatova; A. Ya. Shkuropatov; V. A. Shuvalov (832-840).
The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P+B A − in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P+/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P+B A − stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm−1 reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B A − at 1020 nm is registered in RCs of both mutants. The formation of the B A − band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm−1. Only a partial stabilization of the P+B A − state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P+ and B A − is proposed to explain rapid stabilization of the P+B A − state in native RCs. Small oscillatory components at ∼330–380 cm−1 in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P+B A − free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P+ and B A − is assumed to be another potential contributor to the mechanism of P+B A − stabilization.
Keywords: photosynthesis; charge separation; reaction center; wave packet; electron transfer
Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon by A. V. Surdina; T. I. Rassokhin; A. V. Golovin; V. A. Spiridonova; A. M. Kopylov (841-850).
In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (−82/−20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 ± 43 and 50 ± 24 nM, respectively.
Keywords: ribosomal biogenesis; S7 protein; streptomycin operon; bacteria; translation regulation; regulome; SELEX; SERW
State of oncomarker protein B23/nucleophosmin in HeLa cells by N. M. Vladimirova; N. V. Lobanova; N. A. Potapenko (851-860).
Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.
Keywords: nucleophosmin; monomers; oligomers; HeLa; isoforms; structure
Recombinant maize 9-lipoxygenase: Expression, purification, and properties by E. V. Osipova; I. R. Chechetkin; Y. V. Gogolev; N. B. Tarasova (861-865).
Expression of maize 9-lipoxygenase was performed and optimized in Escherichia coli Rosetta(DE3)pLysS. The purity of recombinant protein obtained during Q-Sepharose and Octyl-Sepharose chromatographies in an LP system at 4°C was >95%. Maximum activity of the lipoxygenase reaction was observed at pH 7.5. Enzyme stability was studied at pH 4.5 to 9.5 and in the presence of different compounds: phenylmethanesulfonyl fluoride, β-mercaptoethanol, ammonium sulfate, and glycerol. HPLC and GC-MS analysis showed that enzyme produced 99% 9S-hydroperoxide from linoleic acid. 13-Hydroperoxide (less than 1%) consisted of S- and R-enantiomers in ratio 2 : 3.
Keywords: maize 9-lipoxygenase; expression of 9-lipoxygenase; chromatography of 9-lipoxygenase; GC-MS analysis; HPLC
Isolation, purification, and study of properties of recombinant hepsin from Escherichia coli by A. A. Raevskaya; E. M. Kuznetsova; M. V. Savvateeva; S. E. Severin (866-872).
A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed.
Keywords: hepsin; proteolytic activity; anthralin; inhibition; cytotoxicity; human prostate adenocarcinoma
Isolation and properties of human transketolase by L. E. Meshalkina; O. N. Solovjeva; Yu. A. Khodak; V. L. Drutsa; G. A. Kochetov (873-880).
Recombinant human (His)6-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).
Keywords: human transketolase; thiamine diphosphate; induced optical activity; circular dichroism spectroscopy
Expression of G-protein coupled receptors in Escherichia coli for structural studies by L. E. Petrovskaya; A. A. Shulga; O. V. Bocharova; Ya. S. Ermolyuk; E. A. Kryukova; V. V. Chupin; M. J. J. Blommers; A. S. Arseniev; M. P. Kirpichnikov (881-891).
To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).
Keywords: autoinduction; expression in E. coli ; GPCR; OmpF; Mistic
Characterization of recombinant fructose-1,6-bisphosphate aldolase from Methylococcus capsulatus Bath by O. N. Rozova; V. N. Khmelenina; I. I. Mustakhimov; A. S. Reshetnikov; Y. A. Trotsenko (892-898).
The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co2+ and inhibited by EDTA. The enzyme displays low K m to fructose-1,6-bisphosphate (FBP) and higher K m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co2+ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C1 assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.
Keywords: fructose-1,6-bisphosphate aldolase; methanotrophs; Methylococcus capsulatus ; Calvin cycle
Conformational changes near the cytochrome P450 active site upon binding of two different ligands by K. N. Myasoedova; K. N. Timofeev (899-904).
It is shown that a stable nitroxyl radical, 4-cyano-2,2,6,6-tetramethylpiperidine-1-oxyl, forms a complex with cytochrome P4502B4 by analogy with the second type substrates by joining directly to pentacoordinate heme iron. The bound radical is inaccessible to water-soluble paramagnetic ions, which confirms its localization in a hydrophobic pocket near the heme. Benzphetamine and N,N-dimethylaniline, the first-type nonpolar substrates, induce conformational changes of the spin-labeled hemoprotein which are evidently accompanied by an increase in the volume of the pocket resulting in emergence of contact with aqueous phase, and the heme-bound spin label becomes accessible to water-soluble paramagnetics. In this case potassium ferricyanide broadens the spin-labeled cytochrome signal and, as a result, lowers the amplitudes of the spectral components. Similar changes were registered at non-micellar concentrations of nonionic detergent Emulgen 913, whose activating effect on hydroxylation reactions is associated, as we showed previously, with its presence in the CYP2B4 active site simultaneously with substrates.
Keywords: cytochrome P4502B4 (CYP2B4); ligands; spin label; EPR spectra; substrates; conformation
Effect of human cell malignancy on activity of DNA polymerase ι by A. A. Kazakov; E. E. Grishina; V. Z. Tarantul; L. V. Gening (905-911).
An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase ι (Pol ι) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol ι preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol ι was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol ι activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol ι in both cases, though to a different extent. In the presence of Mn2+ the Pol ι activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol ι. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.
Keywords: uveal eye melanoma; basal-cell carcinoma; DNA polymerase ι; TLS
Nature of cation-π interactions and their role in structural stability of immunoglobulin proteins by I. A. Tayubi; R. Sethumadhavan (912-918).
Cation-π interactions are known to be important contributors to protein stability and ligand-protein interactions. In this study, we have analyzed the influence of cation-π interactions in single chain immunoglobulin proteins. We observed 87 cation-π interactions in a data set of 33 proteins. These interactions are mainly formed by long-range contacts, and there is preference of Arg over Lys in these interactions. Arg-Tyr interactions are predominant among the various pairs analyzed. Despite the scarcity of interactions involving Trp, the average energy for Trp-cation interactions is quite high. This information suggests that the cation-π interactions involving Trp might be of high relevance to the proteins. Secondary structure analysis reveals that cation-π interactions are formed preferably between residues in which at least one is in β-strand. Proteins having β-strand regions have the highest number of cation-π interaction-forming residues.
Keywords: cation-π; secondary structure; long-range interactions; accessible surface area; stabilization centers; immunoglobulin proteins; structural stability
Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis γ-glutamyl transpeptidase by Hui-Ping Chang; Wan-Chi Liang; Rui-Cin Lyu; Meng-Chun Chi; Tzu-Fan Wang; Kuo-Liang Su; Hui-Chih Hung; Long-Liu Lin (919-929).
The role of the C-terminal region of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581–585, 577–585, 576–585, 566–585, 558–585, 523–585, and 479–585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-γ-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Δ(576–585), pro-Δ(566–585), and pro-Δ(558–585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Δ(577–585), Δ(576–585), Δ(566–585), Δ(558–585), Δ(523–585), and Δ(479–585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and ΔG 0 for the unfolding. BlGGT-4aa and BlGGT sedimented at ∼4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f o) of BlGGT-4aa, BlGGT, Δ(581–585), and Δ(577–585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.
Keywords: Bacillus licheniformis ; γ-glutamyl transpeptidase; C-terminal truncation; autocatalytic processing; analytical ultracentrifugation
Characterization of a new RNase HII and its essential amino acid residues in the archaeon Sulfolobus tokodaii reveals a regulatory C-terminus by Ke Zhan; Zheng-Guo He (930-937).
The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 and 50°C. The activity of wild-type protein was stimulated by Mn2+, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1–195), retained only partial activity, while ST0519 (residues 1–196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short α-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal α-helix is likely involved in the Mn2+-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII.
Keywords: Sulfolobus tokodaii ; RNase HII; nuclease; archaea
Isoforms of human O-GlcNAcase show distinct catalytic efficiencies by Jing Li; Cai-luan Huang; Lian-wen Zhang; Lin Lin; Zhong-hua Li; Fu-wu Zhang; Peng Wang (938-943).
O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63–342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K m value and catalytic efficiency (k cat/K m) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K m value of 0.26 mM and the highest catalytic efficiency of 3.51·103. These results reveal that the N-terminal region (a.a. 1–350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.
Keywords: O-GlcNAcase; isoform; substrate affinity; catalytic efficiency
An easy colorimetric assay for glycosyltransferases by Rui Shen; Shuai Wang; Xiaofeng Ma; Junyang Xian; Jing Li; Lianwen Zhang; Peng Wang (944-950).
Glycosyltransferases are involved in biosynthesis of both protein-bound and non-bound glycans that have multiple and important biological functions in all species. A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. In this work, a novel colorimetric assay for the glycosyltransferase-catalyzed reaction was established. Compared with measuring the newly formed product, which might not exhibit visible absorption, the unreacted acceptor could be readily detected by measuring the visible absorption of the hydrolysis product. In the assay, 4-nitrophenyl-β-D-glycoside (glycosyl-β-pNP) is used as the glycosyl acceptor, which can be hydrolyzed by a special exoglycosidase to release the p-nitrophenol before glycosylation reactions. Absorbance change of the p-nitrophenolate corresponds to unreacted glycosyl acceptor that accompanied the glycosyl transfer. The assay is demonstrated to be useful in the initial characterization of recombinant glycosyltransferases for their kinetic parameters, optimal metal cofactor, and pH value. It provides a simple, sensitive, and quantitative method for assessing glycosyltransferase activity and is thus expected to have broad applications including automated high-throughput screening.
Keywords: glycosyltransferase; colorimetric assay; high-throughput screening