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L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

In mammalian cells, the Golgi apparatus and endoplasmic reticulum have typical structures during interphase: stacked cisternae located adjacent to the nucleus and a network of interconnected tubules throughout the cytoplasm, respectively. At mitosis their architectures disappear and are reassembled in daughter cells. p97, an AAA-ATPase, mediates membrane fusion and is required for reassembly of these organelles. In the p97-mediated membrane fusion, p47 was identified as an essential cofactor, through which p97 binds to a SNARE, syntaxin5. A second essential cofactor, VCIP135, was identified as a p97/p47/syntaxin5-interacting protein. Several lines of recent evidence suggest that ubiquitination may be implicated in the p97/p47 pathway; p47 binds to monoubiquitinated proteins and VCIP135 shows a deubiquitinating activity in vitro. For the cell-cycle regulation of the p97/p47 pathway, it has been reported that the localization and phosphorylation-dephosphorylation of p47 are crucial. In this review, we describe the components involved in the p97-mediated membrane fusion and discuss the regulation of the fusion pathway.

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine spatial relationships between residues on tropomyosin and actin in reconstituted muscle thin filament, and to detect a positional change of tropomyosin relative to actin on the thin filament in the presence and absence of Ca2+ ions. In addition to Cys-190 which is a single cysteine residue in rabbit skeletal muscle alpha-tropomyosin, a new site, Cys-87 which is a unique cysteine residue in a mutant alpha-tropomyosin, was labeled with a resonance energy donor molecule, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS). On the other hand, Gln-41, Lys-61, Cys-374, and the ATP-binding site of actin were selectively labeled with acceptor probes: fluorescein cadaverine, fluorescein 5-isothiocyanate, 4-dimethyl-aminophenylazophenyl 4'-maleimide, and TNP-ATP (or TNP-ADP), respectively. The distances between probes attached to position 87 of the mutant tropomyosin and Gln-41, Lys-61, Cys-374, or the nucleotide-binding site of actin on the reconstituted thin filament in the presence of Ca2+ ion were measured to be 43.2, 49.7, 45.4, and 35.2 A, respectively, and the distance between probes attached to position 190 of tropomyosin and Gln-41 or the nucleotide-binding site of actin were 51.6 and 43.1 A, respectively. The transfer efficiencies between these donor and acceptor molecules were large, so that the efficiency should be very sensitive to changes in distance between probes attached to tropomyosin and actin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The present results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory.

The effects of delta9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]i. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]i induced by 2-arachidonoylglycerol. We also found that 1(3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.

Crystals of a complex of chicken gizzard G-actin and DNase I were soaked in a solution of radioactive 4-hydroxymercuribenzoate (MB). The soaked crystals, which contained 0.93 mol of MB per mol of G-actin, were dissolved in "G-buffer" and digested with trypsin, and the resulting peptides were fractionated by thin-layer chromatography. The MB is exchangeable between peptides that contain cysteine residues, but the data obtained here suggested that MB attached to the cysteine residue at the 373rd position of the G-actin molecule.

Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule. Both are conserved among most members of the cystatin superfamily. We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable [Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K. (1988) J. Biol. Chem. 263, 7655-7659]. Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region. The data indicate the following results. (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin. (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain. (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS)

1. A large fluorescence enhancement of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic alpha-amylase [EC 3.2.1.1] (PPA)-catalyzed hydrolysis of amylose with a number-average degree of polymerization of 16.8. 2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of "multiple attack," which is defined by the number of reattacks on a long chain substrate molecular per one encounter of the enzyme and the substrate. A parameter gamma was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating. 3. The gamma versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism. 4. Based on a reaction scheme involving a "sliding" of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzyme-substrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et. al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule. 5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.

Comparison of the kinetic properties of troponin-C and dansylaziridine-labeled troponin-C revealed that the calcium ion binding and removal reactions with the low affinity Ca2+-binding sites (sites I and II) and the resultant local conformational change are rapid processes, and that the calcium ion binding and removal reactions with the high affinity Ca2+-binding sites (sites III and IV) are also rapid, whereas the resultant local conformational change is a slow process. It is also shown in this work that the latter processes are independent of the former processes.

The kinetics of conformational change of troponin C (TN-C) induced by binding and removal of calcium ions were studied by measuring the fluorescence of tyrosine by stopped-flow spectrofluorometry. When the concentration of free calcium ions in the solution [Ca2+] was increased rapidly from 4X10(-9)M to 1X10(-3)M at neutral pH, a first-order reaction with a rate constant of 13.7 sec-1, which was preceded by a much faster reaction, was observed. In contrast, when [Ca2+] was reduced from 2X10(-3)M to 4.4X10(-8)M, two first-order reactions with rate constants of 7.4 and 0.78 sec-1, preceded by a much faster reaction, were observed.

Induced aggregation of multilamellar liposomes of egg lecithin with several polysaccharides, such as pullulan, dextran, hydroxyethylstarch, and amylopectin, has been investigated in conjunction with structural characteristics of polysaccharides, incubation temperature, ionic strength, and surface charge of liposomes. Aggregation of liposomes as a function of time was followed by monitoring turbidity of the liposome suspension at 600 nm. The inverse relationship between the minimal concentration and molecular weight of polysaccharides for the induced aggregation of liposomes was observed for all the polysaccharides employed. Polysaccharides having larger excluded volume more easily bind to liposomes, leading to the acceleration of the aggregation of liposomes. Hence, the order of efficiency of polysaccharides for the promotion of liposome aggregation was pullulan greater than dextran greater than hydroxyethylstarch congruent to amylopectin. Increasing the surface charge of liposomes brought about a decrease in aggregation irrespective of the sign. Even when negatively charged liposomes were incubated with the cationic DEAE-dextran, the liposome aggregation was depressed. These results including salt effect and temperature dependency of the induced aggregation of liposomes supported a plausible mechanism for the reaction, the polysaccharide-bridging mechanism, which has been provided for the polysaccharide-promoted agglutination of human erythrocytes.

Isethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.

Sulfoacetaldehyde sulfo-lyase, which decomposes sulfoacetaldehyde to sulfite and acetate, was extracted from a bacterium grown on taurine, and purified, and characterized. A method for assay of enzyme activity was devised on formation of a bisulfite adduct with benzaldehyde. The enzyme was purified 14-fold from an extract of cells grown on taurine and appeared homogeneous on disc-electrophoresis. The molecular weight of the enzyme was estimated to be 85,000 by gel filtration. The enzyme required thiamine pyrophosphate (TPP) and Mg2+ for activity and preincubation with TPP and Mg2+ was required for maximum activity. The optimum pH for activity was 7.5. The Km value for TPP was determined to be 2.7 muM and that for sulfoacetaldehyde to be 5.0mM. Sulfite was produced only from sulfoacetaldehyde among a variety of sulfonates tested. rho-Chloromercuribenzoate, EDTA, and sulfite, a reaction product, inhibited the enzyme reaction. The enzyme seemed to be inducible, since activity was found in extracts of cells grown on taurine but not on peptone.

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.

In was shown that the growth of F-actin occurs uni-directionally in vitro; that is, actin monomers (G-ADP-actin) polymerize onto only one of the two ends of a pre-existing seed in the absence of ATP. Fragments of the F-actin-heavy meromyosin complex prepared by sonication were used as seeds. By studying a large number of electron micrographs, we concluded that growth occurs on the tail end of the arrow-head structure of the seed. This result suggests that the thin filament grows in vivo from the center of the sarcomere towards the Z-line. It was further shown that the fragments of F-actin combine with both ends of the seed with nearly equal probability. These results are well interpreted according to the theory developed by Oosawa (1,2).

The midpoint pCa value of the fluorescence titration curve of the complex of 2-[4'-iodoacetamido)anilino)-naphthalene-6-sulfonic acid-labeled troponin (IAANS-Tn) and nonpolymerizable tropomyosin (NPTm) was much larger than that for the complex of Tn containing dansylaziridine-labeled troponin C (DANZ-TnC) and NPTm. The midpoint was pCa 8.25 for the former protein and 6.80 for the latter protein in 0.1 M KCl, 50 mM Na-cacodylate-HCl (pH 7.0); and pCa 7.90 for the former protein and 6.70 for the latter protein in the presence of 3 mM MgCl2 in the same solvent system. The time course of the fluorescence intensity change of the protein complex subsequent to rapid decrease of free Ca2+ concentration of the solution was measured with a stopped-flow spectrophotometer: The process was exponential and its rate constant was 9.9 s-1 for IAANS-Tn-NPTm at pCa 8.95 and 26.6 s-1 for Tn(DANZ-TnC)-NPTm at pCa 8.99 in the absence of MgCl2 in the same solvent system as in the fluorescence titration experiment. IAANS binds to Cys-133 of TnI and DANZ to Met-25 in the low affinity Ca2+-binding sites of TnC. These results suggest that IAANS bound to Cys-133 of TnI does not directly detect the Ca2+-binding to the low affinity Ca2+-binding site of TnC, but does detect the conformational change of the Tn-NPTm complex induced by the Ca2+-binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of skeletal muscle troponin C (TN-C) subunit into skeletal muscle troponin (TN) induces a large increase in the apparent binding constant of Ca2+ to the low affinity Ca2+-binding sites of TN-C (from 1 X 10(5) M-1 to 5.6 X 10(6) M-1 in the presence of 2 mM MgCl2), and a large decrease in the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TN-C (from 230 s-1 to 37 s-1 in the presence of 2 mM MgCl2). On the other hand, no significant modification in the molecular kinetic mechanism of the local conformational change due to the Ca2+ binding or removal reaction with the high affinity Ca2+-binding sites of TN-C is observed as TN-C is incorporated into TN.

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.

The kinetics of the conformational change of troponin-C induced by binding or removal of protons was studied by a stopped-flow pH-jump spectrofluorometric method. In the pH-down experiment (to investigate the kinetics of conformational change from the deprotonated state to the protonated state), a single first-order reaction with a rate constant and amplitude of 1.75-2.4 sec-1 and around 10% respectively, was observed. On the other hand, two first-order reactions with rate constants of 0.84-1.6 sec-1 and 0.08-0.4 sec-1 were observed in the pH-up experiment, the total amplitudes of these reactions being around 10-20%. The pH dependences of the rate constants of these reactions were analyzed in terms of a three-species mechanism.

Sulfite formation from benzenesulfonate was studied in extracts from a bacterium grown on this compound as a main carbon source. The activity of sulfite formation depended on the presence of NADH and oxygen as well as magnesium, suggesting an oxygenation-type reaction. The activity was found in a fraction precipitated by ammonium sulfate at 35-50% saturation; the specific activity was 15 times higher than that of crude extract, probably due to the elimination of inhibitory substances of low molecular weight from the preparation. In a distillate of the reaction mixture, phenol was found. Pyrocatechol as well as benzenesulfonate was oxidized in the crude extract, but phenol was not.

The kinetics of the conformation change of troponin-C (TN-C) induced by magnesium-binding or removal were studied in the absence of calcium ion by measuring the fluorescence intensity change of BIPM bound to TN-C by stopped-flow spectrofluorometry. The kinetic process of the conformational change was biphasic. The rate constants of the two phases were determined as a function of free magnesium ion concentration ([Mg]) of the solution. The [Mg]-dependence of the rate constants was explained by a simple molecular kinetic mechanism: (formula: see text) The dissociation constant of magnesium bound to TN-C was also determined to be 1 x 10(-3) M in the kinetic study.

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.

Static fluorescence titration and fluorescence stopped-flow kinetic experiments o N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide-labeled troponin C showed that the molecular kinetic mechanism of the local conformational change induced by the rapid Ca2+ binding and removal reactions with the high affinity Ca2+-binding sites (sites III and IV) can be explained by the following scheme: [Formula: see text]. Numerical constants in this scheme were determined in this work.

It was found that 6-p-toluidinylnaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to alpha-, beta-, and gamma-cyclodextrin solutions. 2. The following results were obtained by quantitative study of the interactions of three kinds of cyclodextrins with TNS by following TNS fluorescence at pH5.3. and 25 degrees. i) alpha-Cyclodextrin forms a l : l complex with TNS. ii) beta- and gamma-Cyclodextrins form 1 : 1 and also 2 : 1 complexes; in the latter two cyclodextrin molecules bind to one TNS molecule. iii) The dissociation constants of cyclodextrin-TNS complexes were determined to be 54.9 mM for alpha-cyclodextrin, 0.65 mM for beta-cyclodextrin and 0.66 mM for gamma-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin and 32.6 mM for gamma-cyclodextrin. iv)...

The kinetics of conformational change of troponin-C (TN-C) induced by binding or removal of calcium ion were studied in the presence or absence of magnesium ion by measuring the fluorescence of tyrosyl residues by stopped-flow spectrofluorometry. The result was analyzed in terms of first-order kinetics. Two phases were observed both in pCa-up and in pCa-down experiments. The dependence of the rate constants on pCa was explained by a simple mechanism as follows; (see article). The dissociation constants of calcium bound to TN-C, K and K', calculated from the experimentally determined rate constants were K = 3.16 X 10(-7) M, K' = 1.58 X 10(-6) M in the absence of magnesium ion, and K = K' = 1 X 10(-6) M in the presence of 2 mM MgCl2.

Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagent bind, induced by Ca2+ binding to the high affinity Ca2+-binding sites is very different from that induced by Mg2+ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca2+ binding to the low affinity Ca2+-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca2+ binding to the low affinity Ca2+-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca2+ binding to or removal from the high affinity Ca2+-binding sites by following the fluorescence intensity change of ANM-labeled TN-C, respectively. THe kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinty Ca2+-binding sites (rate constants, 3.7-157 s-1) are slower than the kinetic processes associated with the low affinity Ca2+-binding sites (rate constants, equal to or higher than 230 s-1) in the absence of MgCl2. In the presence of 2 mM MgCl2, a new slow phase (rate constant, 10-16 s-1) appears in the kinetic processes associated with the low affinity Ca2+-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl2. Kinetic properties associated with the high affinity Ca2+-binding sites are not essentially altered by addition of 2 mM MgCl2 to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s-1).