Titration of lysine residues on adenine nucleotide translocase by fluorescamine induces permeability transition.

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus.

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

Chemical modification of dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.

The participation of primary amino groups of succinate dehydrogenase in the formation of succinate-Q reductase.

(1) Purified succinate dehydrogenase contains about 49 mol of lysine residues per mol enzyme. Titration of succinate dehydrogenase with fluorescamine indicates that half the lysyl groups are located on the surface of the protein and the other half are buried inside. (2) The reconstitutive activity and the low Km ferricyanide reductase activity of succinate dehydrogenase decreased as the extent of alkylation of amino groups by fluorescamine increased. (3) The inhibitory effects of fluorescamine on both activities are parallel and are succinate concentration dependent. (4) Alkylation of the native succinate-Q reductase by fluorescamine does not affect the enzymatic activity or alter the enzyme kinetic parameters. This indicates that the inhibitory effect of fluorescamine on succinate dehydrogenase is due to the modification of a specific amino group(s) on succinate dehydrogenase which is essential in the interaction with QPs to form succinate-Q reductase. The participation of an ionic group in the formation of succinate-Q reductase supports the idea of the involvement of ionic interaction between succinate dehydrogenase and QPs.

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis.

4-Phenylspiro [furan-2(3H),1-phtalan]3,3'-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions.

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

Fluorescent labelling of proteins of lymphocyte plasma membranes.

Fluorescamine has been used for labelling proteins present on the surface of normal human peripheral blood lymphocytes. Under the conditions of study, 12 labelled proteins could be detected by SDS gel electrophoresis. This method may be of value in biochemical studies of lymphocyte membranes.

The effect of sulfhydryl and amino group reagents on human lymphocyte--sheep erythrocyte rosettes.

We have studied the influence of certain chemical groups, located on the surface of lymphocytes or sheep red blood cells, on the ability of these cells to form spontaneous rosettes. We found that fluorescamine, which reacts with amino groups, inhibits rosette formation, while p-chloromercuriphenylsulfonic acid, which binds to sulfydryl groups, increases the percentage of the rosette-forming lymphocytes. The possible mechanism of action of the above reagents is discussed.

The functional and fluorescence properties of Escherichia coli RNA polymerase reacted with fluorescamine.

1. Fluorescamine (4-phenylspiro[furan-2,(3)1'-phthalan]-3,3'-dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with xi-amino gruops of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning. 2. The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the betabeta' subunits. Holoenzyme and core enzyme show essentially the same behavior. 3. The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected. 4. The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3-8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template. 5. Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4-5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.

Microspectrography of formaldehyde and fluorescamine-induced fluorescence in rabbit pulmonary neuroepithelial bodies: demonstration of a new, probably polypeptide intracytoplasmic substance.

The microspectrographic analysis of the fluorescence emitted by NEB's in gaseous formaldehyde-fixed lung tissue, posttreated with fluorescamine, revealed the presence of numerous primary amino groups which are clearly different from the serotonin identified in our earlier studies and correspond to a new, probably a polypeptide intracytoplasmic substance.

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

Effects of borohydride-treated oligomycins on processes of energy transduction in mitochondria.

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7-keto and the 11-keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP-dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration-linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride-treated antibiotics are also observed in rat-liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4-dinitrophenol), than of Pi formation stimulated by certain amine-fluorescamine modifiers (e.g.) the benzylamine-fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler-stimulated and modifier-stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+-ATPase as a result of hydrogen bonding to, at least, the 7-keto and/or the 11-keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen-bonding is less efficient due to the pronounced directional characteristic of hydrogen-bonding to keto groups.

The inhibition of the calcium transport ATPase of the sarcomplasmic reticulum by fluorescamine: evidence for an oligomeric functional unit of the calcium transport system.

The labeling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluorescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphorylation by [gamma-32P]ATP and [32P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase, it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules.

Activity of phospholipase A2 in the inner membrane of rat-liver mitochondria.

The inner membrane of rat liver mitochondria contains a highly active phospholipase A2 which has alkaline pH optimum and requires Ca2+ in the micromolar range. The phospholipase is particularly active on the endogenous phosphatidylethanolamine and release relatively high amounts of docosahexanoic acid. The phospholipase A2 of mitochondria or mitoplasts is not dependent on calmodulin. Using fluorescamine-labelled mitoplasts there are indications that the enzyme is localized on both sides of the inner membrane.

The influence of enzymatic cleavage and chemical modification of human and rabbit IgG on their reactivity with staphylococcal protein A.

Proteolytic cleavage fragments from rabbit IgG have been isolated and characterized in an attempt to locate the sites involved in the reactivity with Staphylococcal protein A. The plasmin cleavage product Facb together with the pepsin cleavage products F(ab')2 and pFc' failed to react in contrast to the papain Fc fragment. These data, together with data from unfractionated plasmin digests, in which the Facb fragment remains associated with the plasmin pFc' fragment, indicate that inter-domain interactions are important in the maintenance of this activity. beta2-microglobulin was also shown to be unreactive with protein A. Chemical modification studies employing flurescamine, tetranitromethane and potassium cyanate indicate that lysine and tyrosine residues are not involved in the reactivity of human and rabbit IgG with protein A.

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide.

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.

[Ca-ATPase self-fluorescence in the sarcoplasmic reticulum of the rabbit skeletal muscle]. / Issledovanie sobstvennoi fluorestsentsii Ca-ATFazy sarkoplazmaticheskogo retikulma skeletnykh myshts krolika.

The quenching of the intrinsic protein fluorescence of sarcoplasmic reticulum Ca-ATPase from the rabbit skeletal muscles by hydrophylic (NaI, CsCl) or hydrophobic (pyrene, fluorescamine) substances has been studied. CsCl (up to 1 M) has been shown not to affect the intrinsic protein fluorescence while NaI (250 mM) quenches it at 15%, pyrene (8 mkM) decreases the intrinsic fluorescence of Ca-ATPase at 35% and fluorescamine (up to 40 mkM)--at 80%. Possible mechanisms of the interaction of the quenchers with the intrinsic fluorescence of sarcoplasmic reticulum Ca-ATPase are being discussed.