Fluorescent Thiol Reagent Research Articles

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits.

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by Lötscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.

Subunit structure of sesame 13S globulin.

On SDS-polyacrylamide gel electrophoresis, sesame seed 13S globulin was separated into three intermediary subunits termed IS1 IS2 and IS3. Following a treatment with 0.2M 2-mercaptoethanol, the globulin was separated into three acidic subunits termed AS1 AS2 and AS3, and four basic subunits termed BS1 BS2, BS3 and BS4. Two dimensional SDS-gel electrophoresis before and after treatment with 0.2 M 2-mercaptoethanol revealed that IS1 was composed of two combinations of acidic and basic subunits, these being S1 and BS2, and AS2 and BS2. IS2 was found to be composed of AS3 and BS1, and IS3 was composed of AS2 and BS3, and AS2 and BS4. These combinations were consistent with the reactivity of each subunit to a fluorescent thiol reagent. The amino acid compositions were similar among the three acidic subunits and also among the four basic subunits. However, between the acidic and basic subunits, there were great differences in the amino acid composition, especially in the amount of glutamic acid.

Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase

A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcourmarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4′-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4′-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.

Limited proteolysis of a chemically modified third component of human complement, C3, by cathepsin G of human leukocytes.

We have investigated the limited proteolysis of the third component of complement, C3, by a human leukocyte protease, cathepsin G, by using a chemically modified C3, which was prepared by treatment of C3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C3me. Although native C3 was hardly cleaved by cathepsin G, DACM-C3me was cleaved by cathepsin G into three major fragments, which were termed C3c-G (150,000 daltons, 150 kd), C3d-G (25 kd), and C3a-G (10 kd). C3c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C3d-G and C3a-G were single-chain fragments derived from the alpha chain. The N-terminal sequence of C3d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C3d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C3. C3d-G, like C3d-K (a C3d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.

Distribution of sulfhydryl and disulfide groups in ocular tissues.

The localization and concentration of protein-bound sulfhydryl groups and disulfide linkages in mouse ocular tissues were examined with a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM). Corneal epithelium and endothelium, conjunctival epithelium, lens cortex, retinal plexiform layers and inner segment and pigment epithelium were rich in SH groups. Descemet's membrane and lens capsule contained many S-S linkages. Corneal stroma, sclera and retinal outer segment contained a moderate number of S-S linkages. There was a unique distribution of SH groups and S-S linkages in the eye; that is, tissues abundant in SH groups contained few S-S linkages, and tissues abundant in S-S linkages contained few SH groups. The role of SH groups and S-S linkages in the eye was discussed.

Multifunctional cross-linking reagents. I. Synthesis and properties of novel photoactivable, thiol-directed fluorescent reagents.

Bifunctional photo-activable fluorescent thiol reagents of a new type were synthesized. A maleimide group was bonded to an azidocoumarin group via a methylene chain as a spacer. Reagents of this type react first with a cysteine residue of a protein through the maleimide group, and then form another bond with an amino acid side chain of the protein upon irradiation with light, through a nitrene group formed from the azide. Although the reagent is non-fluorescent, the products are highly fluorescent. The fluorescence characteristics of model compounds of these reagents are also described.

Three-dimensional hair follicular differentiation of a trichilemmoma cell line in vitro.

A cell line was established in vitro from a benign hair follicular tumor of human trichilemmoma. Individual and organized cellular differentiation of this cell line was studied. When these cells were cultured for a long time (more than 3 weeks) without subculture, they started to pile up spontaneously. A part of the pile became indented and simultaneously the opposite side of the indentation budded out. The bud slowly elongated 2 to 3 mm in length in 8 to 12 weeks in culture. Light and electron microscopy revealed the internal structure of piles and elongated buds to be a three-dimensional hair follicular structure. The cells in the outermost layer were least mature. These were cuboid in shape and contained glycogen. The cells in the middle layer were more differentiated with a decreased amount of glycogen and an increased number of tonofilaments and desmosomes. The cells in the innermost layer were most differentiated. Cells were flat in shape and highly convoluted. The cell membrane was thickened as observed in cornified cells in vivo. These organized differentiations were also confirmed by histochemical and immunocytochemical studies; using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide method, free sulfhydryl groups were detected but disulfide bonds were absent in the early cell culture. Disulfide bonds increased slowly and accumulated in the innermost layer of piles. Accumulation of keratin substances, detected by indirect immunofluorescence method using anti-human keratin antibody, was also observed specifically in the piles. These results suggest that an established cell line of human trichilemmoma spontaneously produced, without stromal influence, hair follicular structures as well as individual cell differentiations in vitro as do trichilemmal (hair follicular) cells in vivo.

Characterization of the uptake and toxicity of a fluorescent thiol reagent.

3-(4-Maleimidylphenyl)-4-methyl-7-diethylamino coumarin (CPM), is a fluorescent thiol-binding agent. CPM is nontoxic to Chinese hamster V-79 cells (2 x 10(5) cells/ milliliter) exposed to 2.5 micrograms/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drug:cell ratio. Using flow cytometry, cellular binding of CPM correlated with inhibition of DNA synthesis. In cells pretreated with the thiol binding drugs N-ethylmaleimide, diamide and diethylmaleate or the carcinogens 4-nitroquinoline-1-oxide and AF-2 (furylfuramide), the subsequent binding of CPM was reduced by 40% or more.

N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM): an alternative label for fluorescence tracing

N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), a fluorescent thiol reagent, has a high molar extinction coefficient and a high quantum yield when it reacts with protein sulphhydryl bonds. DACM proved to be effective as a fluorescent probe when used for labelling rabbit immunoglobulin G anti-human γ-chain (IgG). DACM-labelled rabbit immunoglobulin G anti-human γ-chain conjugates (DACM-IgG) can be purified efficiently by Sephadex G-50 gel chromatography. The immunological properties of the IgG are not altered appreciably by conjugation. As a label DACM has advantages with regard to ease of preparation, specific immunofluorescence and strong photoresistance.

Two sulfhydryl groups near the active site of thiolase I from porcine heart: modification of thiolase with the fluorescent thiol reagent S-mercurio-N-dansyl-L-cysteine.

Two sulfhydryl groups near the active site of thiolase I from porcine heart: modification of thiolase with the fluorescent thiol reagent S-mercurio-N-dansyl-L-cysteine.

Sulphhydryl crosslinking in cutaneous apoptosis: a review.

Histochemical findings of cutaneous apoptosis are reviewed with special reference to the sulphhydryl groups. Using stain techniques with the new fluorescent thiol reagent, N‐(7–dimethylamino‐4–methylcoumarinyl) maleimide (DACM), it was shown that sulphhydryl crosslinking occurs in colloid bodies and sunburn cells and also participates, at least in part, in amyloid formation. As the DACM staining clearly demonstrated the localization of sulphhydryl and disulphide groups in the epidermis, this technique can be of help dealing with the problem of the origin of cutaneous apoptotic bodies.

A fluorometric method for determination of C3b inactivator

A simple method for the determination of C3b inactivator in human plasma was investigated. Fluorescent-labeled C3, which was prepared by treatment with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide, was used as the substrate. Diluted human plasma is incubated with fluorescent C3, reduced with 2-mercaptoethanol, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The fluorescent cleavage product is then determined by scanning the gels with a fluorospectrophotometer. This method is simple and sensitive enough to determine C3b inactivator activity in 1 μl of human plasma.

A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum.

The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.

Sulphhydryl and disulphide stainings of amyloid: a comparison with hyaline body

Histochemical staining for sulphhydryl (-SH) groups and disulphide (S-S) bonds was undertaken in 10 cases of cutaneous amyloidosis using the new fluorescent thiol reagent. A weak S-S fluorescence (but not -SH) was detected in dermal amyloid, suggesting that cross-linking of -SH groups may occur in amyloidogenesis. Since in normal skin S-S bonds were stained only in the horny layer and negligibly in the dermis, our findings seem to support the view that amyloid is derived at least partially from epidermal cells.

Sulphhydryl and disulphide stainings of subepidermal hyaline bodies.

The localization of sulphhydryl (-SH) groups and disulphide (-S-S-) bonds in subepidermal hyaline bodies was examined using histochemical techniques with the specific fluorescent thiol reagent (DACM). In lichen planus, hyaline bodies showed -S-S- but not -SH staining, suggesting that erythematosus and dermatomyositis, there were not only -S-S-positive hyaline bodies, considered of epidermal origin, but also -S-S- negative ones. The latter may consist of dermal components, since no dermal components are stainable with DACM. DACM staining therefore seems to be of help in determining the origin of hyaline bodies.

Histochemical staining of sunburn cells for sulphhydryl and disulphide groups: a time course study.

Ultraviolet light (UV)-exposed rabbit skin was stained with a fluorescent thiol reagent or haematoxylin and eosin to investigate a time relationship between histochemical stainings for sulphhydryl (-SH) and disulphide (S-S) groups and sunburn cell (SBC) production. SBCs appeared by 8 h after irradiation, increased and showed a peak at 48 h, then declined to zero by 96 h. SBCs showing -SH staining increased to a maximum of 41--59% at 24--48 h and declined to 26--45% by 72 h, whereas S-S-stained SBCs were only a few at 8--24 h and then increased up to 65--85% by 72 h. The results seem to indicate that S-S linkage may occur in the process of SBC production.

Involvement of 17K dalton light chain of smooth muscle myosin in substrate-induced conformational change.

Conformational changes of the region involving 17K dalton light chain (G2) in gizzard myosin induced by ATP and its analogs were studied by a combination of light chain exchange and fluorescence spectroscopy. The cysteinyl residues of myosin light chain mixture, 20K dalton G1 and 17K dalton G2 chain, were labeled with a fluorescent thiol reagent, N-(1-anilionon-aphthyl-4ymaleimide (ANM). Chicken gizzard myosin was incubated with a large excess of the fluorescence labeled exogenous light chains in 2 M urea. After removal of urea and unbound light chains, a strong fluorescent band was observed at the position of the 17K dalton light chain (G2) of myosin on SDS PAGE. There was no difference in enzymatic activity and light chain composition between myosins before and after light chain exchange. The emission spectrum of ANM label in 17K dalton light chain in the bound state in myosin showed a fluorescence enhancement and a blue shift on adding ATP but not ADP. The spectral change disappeared after conversion of ATP to ADP. Various nucleoside triphosphates other than ATP had similar effects on the fluorescence spectrum. The relative effectiveness of most nucleotides on the fluorescence spectral change of ANM in the light chain bound in myosin agreed well with that on tryptophan residues in myosin, except in the case of ADP and CTP. These results suggest that 17K dalton light chain of gizzard myosin is closely related to the ATPase site of myosin.

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.

The fluroescent profiles of keratohyalin granules of newborn rat epidermis with a new fluorescent thiol reagent (DACM)

A highly specific fluorescent thiol reagent, N-(7-Dimethylamino-4-methyl-coumarinyl)maleimide (DACM) has been used to examine the newborn rat skin in order to elucidate the cystine-rich keratohyalin granules or dense homogeneous deposits. After reduction of the disulphide bond with 2-mercaptoethanol, the periphery of the stratum corneum cells showed a very distinct fluorescence and the cytoplasm of the stratum corneum, granulosum and spinulosum showed moderate fluorescence. No fluorescence was observed, however, over the keratohyalin granules themselves. Intense, spotty fluorescence was observed from the remnants of nuclei and at the periphery of large keratohyalin granules of the stratum granulosum cells, which were assumed to be dense homogeneous deposts according to their distribution pattern. As the intense fluorescence elicited with DACM determines the abundance of cystine content in the material, cystine was found to be very scarce in keratohyalin granules, but abundant in dense homogeneous deposits and at the periphery of the stratum corneum cells.