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.     

Effect of nonionic detergents on the activity of a thermostable lipase from Bacillus stearothermophilus MC7

The influence of nonionic surfactants on the activity of a novel thermostable lipase from Bacillus stearothermophilus MC7 was investigated with a view to its potential for synthesis of structured lipids. A large number of modifiers within a broad concentration range were applied. The activity of the enzyme was measured at a relatively high reaction temperature. Highest degree of activation was observed when PEG₆₀₀₀ was applied (up to 2.3-fold increase). Modification essentially changed the performance of the lyophilised preparations—they keep up to 80% of the activity of the native enzyme in the presence of a detergent against 30% in its absence. The effect of sorbitan esters (spans) and polyoxyethylene derivatives of sorbitan esters (tweens) on lipase MC7 was estimated, their HLB value varying within the interval 2.1–16.7. Tweens were strong inhibitors at higher concentrations. For all spans, excepting span 60, an increase of enzyme activity with concentration was observed. All studied additives slow down the process of thermal denaturation. Lipase preparations preserve more than 60% of their activity after 30-min incubation at 75 °C in the presence of tween 60 or PEG₄₀₀₀.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

Acute and chronic effects of an anionic surfactant on some freshwater tubificid species

We report the results of research on acute and chronic effects of linear alkylbenzensulfonate (LAS) on two tubificid species. 96 h LC₅₀ assay values were estimated at 10° for Limnodrilus hoffmeisteri and Branchiura sowerbyi exposed to different concentrations of LAS dissolved in water, both with and without sediment. The presence of sediments modified LAS toxicity and increased values: NOEC and LOEC resulted in values 2.5 times higher for Branchiura sowerbyi and 4–4.5 times for Limnodrilus hoffmeisteri, when the sediments were present. The chronic effects induced by a long exposure to LAS were evaluated for different stages of the biological cycle of Branchiura sowerbyi. Using concentrations between the NOEC and LOEC (0.5, 2.5, and 5 ppm), with control, we could observe that: 1) at 5 ppm the cocoons were laid precociously compared to controls, 2) in all treated series the number of cocoons was lower than in controls, 3) the mean number of oocytes per cocoon was lower for the worms submitted to LAS, compared to the control, 4) the period of embryonic development was similar for all used concentrations and for control, and 5) the number of degenerated cocoons was unchanged by the LAS treatment.     

Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.Abbreviations A.PRX anionic peanut peroxidase - C.PRX cationic peanut peroxidase - PcAb polyclonal antibodies - ELISA enzyme-linked-immuno-sorbent-assay - TFMS trifluoromethane sulfonic acid     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

蚕豆叶肉原生质体电融合的研究

本文研究了蚕豆叶肉原生质体经透明质酸酶、核糖核酸酶、神经氨酸酶、碱性磷酸酶、胰蛋白酶、脂肪酶六种水解酶和SDS、Triton X-100、CTMAB三种表面活性剂以及秋水仙素、细胞松驰素B处理后的电融合过程。结果表明:胰蛋白酶处理后的原生质体融合率明显下降;碱性磷酸酶、脂肪酶以及核糖核酸酶、透明质酸酶、神经氨酸酶处理的原生质体电融合率均有不同程度的上升。Triton X-100和CTMAB促进原生质体的电融合,但较高浓度(0.01%)的SDS起抑制作用。秋水仙素和细胞松驰素B处理的原生质体其电融合率有较大幅度的增高。     

Effect of concentration and environmental form of tetradecenyl succinic acid on its mineralization in soil

Tetradecenyl succinic acid (TSA) is the major component of a detergent builder (C12-C14 alkenyl succinic acid), which is inherently biodegradable. ¹⁴C-TSA was dosed as a component of sewage sludge into a soil with a history of sludge amendment at final added concentrations of 1.5 and 30 mg (kg soil)^-1. In addition, it was dosed to the soil in an aqueous solution to a final added concentration of 30 mg (kg soil)^-1. Dose and form were found to have a pronouced effect on the mineralization kinetics. When dosed in a realistic form and concentration (i.e. 1.5 mg (kg soil)^-1 as a component of sludge), TSA was mineralized at its highest rate and to its greatest extent, and the mineralization half-life was 2.4 days. When dosed at 30 mg (kg soil)^-1 as a component of sludge, mineralization began immediately, and the half-life was 23 days. In contrast, when dosed at this concentration in aqueous solution, the onset of mineralization was preceded by a 13 day lag period and the mineralization half-life was 69 days. Primary biodegradation and mineralization rates of TSA were very similar. Approximately, half the radioactivity was evolved as ¹⁴CO₂, while the remaining radioactivity became non-extractable, having presumably been incorporated into biomass or natural soil organic matter (humics). This study demonstrated that TSA is effectively removed from sludge-amended soils as a result of biodegradation. Furthermore, it showed the effect that dose form and concentration have on the biodegradation kinetics and the importance of dosing a chemical not only at a relevant concentration but also in the environmental form in which it enters the soil environment.     

Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 Å resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (T II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 Å. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed. © 1994 Wiley-Liss, Inc.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem