Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.     

Liposome-based homogeneous luminescence resonance energy transfer

There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor-ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin-streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.     

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.     

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

A solid-phase assay for identification of modulators of prion protein interactions

The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K-resistant form of the prion protein, which has been converted from the endogenous, proteinase K-sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high-throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and the protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a nonisotopic labeling strategy for large-scale screens.     

Biotin chemoresponse in Paramecium

Paramecium tetraurelia locate their␣foodsource by detecting bacterial metabolites and altering swimming behavior to congregate near bacterial populations on which they feed. Several attractants, such as folate, glutamate, cAMP and acetate have been identified and various aspects of chemoreception, signal transduction and effector mechanisms have been described. Here we characterize the Paramecium chemoresponse to biotin. An essential enzymatic cofactor in all cells, biotin is secreted by a large number of bacterial species during growth phase. P. tetraurelia are strongly attracted to biotin with a half-maximal behavioral response at 0.3 mmol · 1⁻¹ in T-maze assays. Physiological recordings from whole cells show that cells hyperpolarize in a concentration-dependent manner in biotin. Whole-cell binding assays utilizing ³H-biotin identify a saturable and specific binding site with an apparent dissociation constant of 0.4 mmol · l⁻¹. The biotin analogs desthiobiotin and biotin methyl ester are also strong attractants. Diaminobiotin fails to attract P. tetraurelia at 1 mmol · l⁻¹, but does interfere with the biotin chemoresponse and displaces ³H-biotin from whole cells. We hypothesize that the keto group and/or fidelity of the ureido ring of biotin are necessary for biotin chemoresponse. Accepted: 23 April 1998     

地噻咪松诱导乳鼠胸腺细胞凋亡的研究

本文用透射电镜的方法,研究了地噻咪松诱导乳鼠胸腺细胞的凋亡,并确立了乳腺胸腺细胞中凋亡细胞学形态的特征;含溴化乙锭的琼脂多糖电泳检测乳鼠胸腺细胞中凋亡细胞的断裂ＤＮＡ;末端标记法直接,持异的标记乳鼠胸腺细胞中凋亡细胞断裂ＤＮＡ。     

白色念珠菌摄取生物素的机制研究

本文进一步实验显示处于生物素饥饿状态的白色念珠菌摄取生物素具可饱合性和特异性。从饱合曲线测算出主动摄入生物素的半数最大初速率（１／２Ｖｍａｘ）所需生物素浓度,即Ｋｍ值为０７９±０１１（平均数±标准差,ｎ＝８）;叠氮钠（５ｍＭｏｌ／Ｌ）处理后,其Ｋｍ值增到２３９±０６４μｍＭｏｌ／Ｌ（平均数±标准差,ｎ＝３）。生物素之结构同源分子,如脱硫生物素、生物胞素、甲基酯生物素和对硝基苯酯生物素等竞争性抑制其主动摄取生物素,但是２亚氨基生物素却完全无抑制作用。在生物素摄入充足的白色念珠菌（在含２０ｎＭｏｌ／Ｌ生物素的培养基中生长）,生物素主要通过简单的弥散方式进入菌体内。由于血清中的生物素含量在ｐＭｏｌ／Ｌ到几个ｎＭｏｌ／Ｌ之间,白色念珠菌在体内可能通过主动摄取生物素机制满足其营养代谢需求。本研究为发展抗白色念珠菌新举措可能提供某种理论基础     

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

We have reported that production and characterization of antibodies highly specific to 5-methyl-cytosine (5mC) and the development of a sensitive immunochemical method for the detection of 5mC in DNA [FEBS Lett. (1982) 150, 469]. Extension of this method to two other modified bases, 6-methyladenine (6mA) and 7-methylguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species.