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.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Localisation autoradiographique des ions Cl− et Na+ dans les cellules à chlorure de la branchie d'anguille (Anguilla anguilla L.) adaptée à l'eau de mer

Résumé Nous avons montré par une technique autoradiographique que les ions Cl^– et Na⁺ sont concentrés dans les cellules à chlorure de la branchie d'anguille adaptée à l'eau de mer. La signification de cette accumulation ionique plus marquée vers le pôle apical de ces cellules a été discutée par rapport à l'excrétion branchiale de ces ions en eau de mer.

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Autoradiographic localization of Cl^– and Na⁺ ions in the chloride cells of sea water adapted eel (Anguilla anguilla L.) gills

Summary With an autoradiographic technique Cl^– and Na⁺ ions have been shown to be localized in the chloride cells of sea water eel gills. The significance of this accumulation, more marked towards the apical pole of these cells, is discussed with regard to branchial excretion of these ions in sea water.

     

A transient voltage-dependent outward current in cultured cerebellar granules

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of –80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was –77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of –78 mV and completely inactivated with –50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotians tabacum: spatial relationship proposed between resulting electrical fields and phytophthoran zoospore infection

Abstract Growing roots of Nicotiana tabacum var. Havana generate transcellular ion currents which traverse developing and wounded tissues. Positive current of around 10 mA m^?2 enters meristematic and elongating cells at the tip of primary roots. The growing tips of first order laterals are also traversed by a similar positive current with a density of around 2.0 mA m^?2, as are immature laterals emerging at the primary root surface. These self-generated ion currents flow basipetally through developing tissues and leave from mature non-elongating tissue. A large positive current of around 70 mA m^?2 also enters induced wound sites on the primary root surface. Motile zoospores of the fungal pathogen Phytophthora parasitica var. nicotianae have been reported to associate preferentially with these regions of the root. This might suggest that electrotaxis may be part of the mechanism by which zoospores locate root regions susceptable to fungal infection.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

Measurement of magnetically induced electric fields in conductive media near a 60 Hz current-carrying wire.

Electric fields induced in a conductive body by the magnetic field of a current-carrying wire were analyzed theoretically and experimentally to assess the dosimetric importance of highly nonuniform, field-exposure conditions. Experimentation revealed that a 60-Hz magnetic field was inversely proportional to the radius of a wire bundle carrying 100 A within a 0.5-m2 test area. A miniaturized electric field probe was used to measure the electric fields induced in 5-cm-deep, saline-filled models. In the theoretical analysis, numerical estimates of induced fields were made by a spreadsheet method. The theoretical calculations and the measured values of induced electric fields were generally in good agreement. The induced fields were in a plane perpendicular to a vertically incident magnetic field; the maximally induced fields were in areas nearest the wire bundle. The strength of the induced field increased with model size: from 96 microV/cm in a 10 x 10 cm model to 176 microV/cm in a 40 x 40 cm model. The strength of the field induced in a 20 x 20 cm model decreased with increasing model-to-wire spacing: from 132 microV/cm for a 1-cm spacing (2-mT maximum, incident field) to 50 microV/cm for a 6-cm spacing (0.33-mT maximum). The results indicate that increases in local values of nonuniformly incident fields produce relatively small increases in induced electric fields. This finding may be important in dosimetric consideration of circumstances, such as use of electric blankets, in which fields of low average strength are accompanied by intense local fields.