Takamitsu Sekine

The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.

Fluorescent thiol reagents: XII. Fluorescent tracer method for protein SH groups using N-(7-dimethylamino-4-methyl coumarinyl) maleimide. An application to the proteins separated by SDS-polyacrylamide gel electrophoresis

Abstract A new fluorescent thiol reagent, N -(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), had a high molar extinction coefficient and a high quantum yield when reacted with protein SH groups. We could see directly the position of the labeled proteins in the polyacrylamide gel under ultraviolet illumination, since the maximum emission wavelength of DACM was about 480 nm. In addition, the quantum yield of DACM attached to the denatured proteins was almost identical regardless of the protein species. The properties enabled us to measure by fluorometry the amount of DACM, on a picomole level, incorporated in each protein band separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Fluorescence and structures of proteins as measured by incorporation of fluorophore: IV. Synthesis and fluorescence characteristics of N-(p-(2-benzimidazolyl)phenyl) maleimide

Abstract 1. 1.|A sulfhydryl reagent N-(p-(2- benzimidazolyl)phenyl ) maleimide (BIPM) was synthesized and the fluorescence characteristics of its derivatives were studied. 2. 2.|The fluorescence behavior agrees with an empirical rule that maleimide derivatives are practically nonfluorescent, while their addition products with sulfhydryl compounds are fluorescent. 3. 3.|BIPM may be employed as a tracer of SH-containing peptides in structural studies of proteins. Further possible applications are discussed.

Fluorescent thiol reagents. VI: N-(1-Anilinonaphthyl-4)maleimide; a fluorescent hydrophobic probe directed to thiol groups in protein☆☆☆

N-(1-Anilinonaphthyl-4)maleimide (ANM) (m.p. 207-208.5 degrees C), nonfluorescent, reacts selectively with thiols to give addition products which are strongly fluorescent (excitation maximum 355 nm, emission maximum 448 nm, in ethanol). 0.7 mole of ANM was introduced into thiol groups of egg albumin without modifying any other amino acid residues. In the fluorescence spectra of the reaction products of ANM with thiols, the quantum yields increase and the emission maxima shift towards the blue as the solvent polarity decreases. This remarkable solvent dependence was described in terms ofZ value of the solvents. ANM is thus expected to be a useful fluorescent hydrophobic probe directed to thiol groups in protein.

Fluorescent thiol reagents. V. Microfluorometry of thiol compounds with a fluorescent-labeled maleimide.

Abstract 1. 1. N-(p-(2-Benzimidazolyl)phenyl)maleimide (BIPM) has been used for the quantitative determination of thiol compounds. The method is based on the particular fluorescence characteristics of this reagent: its adducts to thiol compounds have strong fluorescence (λmax, ex 315 mμ, λmax, em 365 mμ), while the reagent itself has no appreciable fluorescence. 2. 2. The addition reaction of 2 × 10−6M BIPM to thiols is completed within 30 min at pH 6.85 and 0°C. A linear relation was observed up to 10−5M of the reagent between concentration of adduct and intensity of fluorescence. 3. 3. A very sensitive fluorometric determination of thiol compounds has been developed based on the above results. The detection limit was 10−6M concentration of thiols, with 0.1 ml of sample, corresponding to 0.03 μg reduced glutathione, with an accuracy of 3%. 4. 4. Using the fluorometric method, the oxidation rate of glutathione catalyzed by metal ions was examined and the contents of thiols in human lacrimal fluid and seurm were determined.

Preparation and properties of smooth muscle myosin from horse esophagus.

Abstract Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle. The ratio of the absorption of myosin S at 280 nm to that at 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/10 5 g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KCl and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A. Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per 10 5 g of protein as horse and rabbit skeletal myosin A (about 8 moles/10 5 g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline. Ca 2+ -ATPase of myosin S in the presence of 0.5 M KCl and Mg 2+ -ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca 2+ -ATPase in the presence of 0.05 M KCl were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25°, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N -ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca 2+ -ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca 2+ -ATPase during the course of the reaction of sulfhydryl groups of myosin with N -ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A.

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil α-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified1 Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP)2. Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents3. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings4,5 that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate6 and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry: II. Effect of tropomyosin, troponin and calcium ion on conformation of anilinonaphtylmaleimide-labeled F-actin

Abstract F-actin labeled with N-(1-anilinonaphthyl-4) maleimide, an SH-directed fluorescent probe, was used to analyse the transmission of the effect of Ca2+ from troponin to F-actin. 1. 1.|The fluorescence emission maximum wavenumber of anilinonaphthyl-maleimide-labeled F-actin (2.29 · 104 · cm−1-2.27·104 cm−1) indicated that hydrophobicity of the microenvironments around the thiol(s) bound with the dye is 72–76 in terms of the Z value. The emission maximum did not change by combination of the actin with tropomyosin or tropomyosin-troponin complex in the presence and absence of Ca2+, but the quantum yield and fluorescence lifetime significantly changed. The lifetime of the labeled F-actin-tropomyosin complex was restored to the level for F-actin-tropomyosin complex by adding 10−5 M CaCl2, but recovery of the quantum yield was incomplete. 2. 2.|The apparent Ca2+-binding constant obtained from the change in fluorescence lifetime, 1.3·106 M, was in good agreement with the reported values obtained from different methods. 3. 3.|In isothermal experiments, no viscosity dependence of the fluorescence polarization, P, was detected for F-actin and all its complexes. 4. 4.|Thermal dependence of P revealed that a thermally activated portion involving the fluorescently labeled thiol(s), the rotational relaxation time of which was about 40 ns, appeared on addition of tropomyosin. A thermal transition in conformation around the labeled region was also observed on adding tropomyosin, with the transition temperature of 31 °C. 5. 5.|On addition of Ca2+ to the F-actin-tropomyosin-troponin system, the thermally activated portion and the thermal transition in conformation completely disappeared. Based on the results mentioned above, the conformation changes at the labeled region are discussed in relation to the transmission of Ca2+ effect on troponin to actin.

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide.

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.