Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.     

Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

γ-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.     

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

Rabbit liver transglutaminase: physical, chemical, and catalytic properties.

Transglutaminase (R-glutaminyl-peptide:amine alpha-glutamyl-yltransferase [EC 2.3.2.13]) has been purified to apparent homogeneity from extracts of rabbit liver. The enzyme is a single polypeptide chain of approximately 80 000 molecular weight containing one catalytic site per molecule. That the isolated enzyme is the rabbit counterpart of the well-characterized guinea pig liver transglutaminase is evidenced by the similarities in their amino acid compositions and in their enzymic activities toward several substrates, together with the fact that the isolated rabbit enzyme is immunologically distinct from both rabbit plasma and rabbit platelet blood coagulation factor XIII. A striking difference between the catalytic activities of the rabbit and guinea pig enzymes is the low activity of rabbit transglutaminase for hydroxylamine incorporation into benzyloxycarbonyl-L-glutaminylglycine, a reaction for which the guinea pig enzyme shows a high reactivity. This finding reveals the cause of error in an earlier report (Tyler, H.M., and Laki, K. (1967) Biochemistry 6, 3259) that rabbit liver contains little, if any, of the enzyme. Preparation of, and analytical data on, several glutamine-containing peptide derivatives used in this study are reported here.     

Natural occurrence of zearalenone in rice and soybean produced in Korea

88 rice and 75 soybean samples were collected from 8 provinces of Korea from March through September in 1988. The Fusarium mycotoxins, zearalenone was analyzed by direct competitive enzyme linked immunosorbent assay. 10.2% of rice and 9.3 % of soybean samples contained detectable zearalenone. The average levels of zearalenone of rice and soybean samples were 11.78μg/kg and 7.70μ/kg, respectively.