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Large amounts of Ca2+ (almost 20 nmol/108 cells) are released from platelets by exocytosis, This secretory-granule-associated Ca2+ does not contribute to the cytosolic free Ca2+ ([Ca2+]i), which is controlled by the much smaller agonist-sensitive Ca2+ pool, unless high (1 μM), but not low (0.04 μM) concentrations of ionomycin are present. Low concentrations of ionomycin release Ca2+ almost exclusively from the agonist-sensitive stores. In aspirinated platelets incubated in the presence of 0.5 mM EGTA the extensive depletion of the agonist-sensitive stores is obtained by the combined action of low ionomycin and the endomembrane Ca2+-ATPase inhibitor thapsigargin (which individually promote only a partial depletion). The subsequent decay of [Ca2+]i is increased by phorbol-myristate acetate, confirming that Ca2+ efflux from platelets is potentiated by the activation of protein kinase C [Pollock, W. K, Sage, S. O. & Rink, T. J. (1987) FEBS Lett. 210, 132- 140]. A novel type of control of Ca2+ efflux appears to be exerted by the filling state of the stores. Treatment with low ionomycin or thapsigargin determines the release of a fraction of the stores- associated Ca2+ : the subsequent decay of [Ca2+]i is slow. The decay rate of [Ca2+]i accelerates after extensive depletion of the stores following the addition of thapsigargin or ionomycin. If the depletion of the stores is induced by thrombin, added alone or in combination with thapsigargin, the increases of [Ca2+]i are the same and the subsequent decay rates are largely superimposable; however a large fraction of [Ca2+]i is reaccumulated into the stores in the absence, but not in the presence of thapsigargin, indicating that Ca2+ efflux is activated when the stores are empty. Ca2+ efflux can proceed against a concentration gradient. In 45Ca-loaded platelets, the thrombin-promoted 45Ca efflux is potentiated by thapsigargin. The protein-kinase-C-dependent and store-depletion-dependent stimulations of 45Ca efflux are additive. These observations indicate that, in addition to being activated by protein kinase C, Ca2+ efflux from platelets is activated by the depletion of the stores. The two activations appear to be additive. [ABSTRACT FROM AUTHOR]

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopynivate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurocheroical function to be investigated further. [ABSTRACT FROM AUTHOR]

The inactivation of aspartate aminotransferase in the course of the incubation with L-serine O-sulfate is protected by thiosulfate. It has been found that also L-cysteine sulfinic acid is an inactivator when it is incubated with the enzyme, while L-serine-O-phosphate and L-cysteine-S-sulfonate are inactive. The inactivation by L-cysteine sulfinic acid is increased by the simultaneous addition of 2-oxoglutrate and is protected by thiosulfate. Cysteine-S-sulfonate has been detected in the incubates of the enzyme with L-serine-O-sulfate protected with thiosulfate. It has been concluded that the protection by thiosulfate is due to the β-addition of thiosulfate to the inactivator aminoacrylic acid. The resulting cysteine-S-sulfonate thus represents an inactive dead-end product formed in the course of the protection by thiosulfate. It is suggested that this reaction could play the role of protecting enzymes sensitive to aminoacrylic acid and of inserting inorganic sulfur into aminoacrylic acid, producing a cysteine derivative. [ABSTRACT FROM AUTHOR]

Intracellular expression of recombinant Schistosoma mansoni protein p28 (Smp28) in soluble form to a concentration of more than 6 g/l culture in Saccharomyces cerevisiae was accompanied by a posttranslational modification, which occurred during the late stage of the culture. The modified protein, which had a reduced isoelectric point, was isolated by anion-exchange HPLC and characterized by tryptic mapping by means of on-line reversed-phase HPLC/electrospray mass spectrometry. Comparison with non-modified recombinant Smp28 allowed us to localize the modification to the N-terminal hexapeptide AGEHIK, which had an increased mass of 26 Da. Reversed-phase HPLC of the modified peptide with a shallow acetonitrile gradient revealed the presence of two components of identical mass and amino acid composition. Both peptides were inaccessible to N-terminal Edman sequencing, indicating that a rearrangement of the N-terminal region of recombinant Smp28 had taken place during tryptic digestion lending to two isomeric, N-terminally blocked peptides. Deuterium-exchange mass spectrometry showed that the modified peptides lacked two exchangeable protons, suggesting cyclic modifications implying the N-terminal amino group. Tandem mass spectrometry by means of the nano-electrospray technique and collision-induced dissociation allowed us to identity the modified sites as Ala 1, His4 and Lys6 based on a characteristic modified a1 ion of Ala1 (70.0 Da), a modified immonium ion of His4 (136.0 Da) and a modified y1″ ion (173.2 Da) of Lys6. Combination of all the above results led to the conclusion that recombinant Smp28 was initially modified at its N-terminus by addition of acetaldehyde to form an aldimine which rearranged during tryptic digestion to two different cyclic peptides. [ABSTRACT FROM AUTHOR]

Guinea-pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20-30 pmol min-1 mg protein-1. A transient increase of the H2O2 release is induced by the addition of i mM Ca2+, which declines within 60-90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40-100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min-1 mg-1) upon depletion of the synaptosomal glutathione by treatment with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+-induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min-1 mg-1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCl or with veratridine. [ABSTRACT FROM AUTHOR]

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i. e. L-Cystathionine, L-lanthionine and s-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible rote of this enzyme in their biosynthesis. [ABSTRACT FROM AUTHOR]

An enzyme which actively transaminates L-cystathionine, L-cystine, L-lanthionine and S-aminoethyl-L-cysteine has been purified from bovine kidney. The transaminase appears to be pure up to 90% and probably consists of two subunits of similar molecular mass of about 47 kDa. The enzymatic products arising from the transamination of L-cystathionine and related compounds spontaneously cyclize into ketiminic structures, which are the immediate precursors of unusual imino acids recovered in biological materials. The specificity towards other amino acid and oxo acid acceptors is similar to the specificity exhibited by rat kidney glutamine transaminase. This suggests that the sulfur amino acid transaminations that have been described could be performed by the bovine kidney glutamine transaminase. [ABSTRACT FROM AUTHOR]

The heterogeneity of crystalline rhodanese from bovine liver has been studied. The enzyme was separated by preparative disc electrophoresis into four stable forms designated 1(3.5 %), 11(17.0%)' 111(62.0%) and IV (17.5 %). All these components have the sante molecular weight of about 35000 both in native and denaturing conditions. No differences in the primary structure have been detected in amino acid composition and by peptide mapping. Although the isolated forms show similar values of specific activity, except form I, their content of transferable sulfur is different. The enzyme-sulfur complexes of the four components have slightly different circular dichroism spectra in the near ultraviolet, though their spectra in the sulfur-free state are identical. We conclude that these forms are conformational isomers originated from form IV which is predominant in the mitochondrial extract of liver cells. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate and other aromatic aldehydes inactivate rhodanese. This inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5′-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5′-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate—rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5′-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship between loss of activity and pyridoxal 5′-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5′-phosphate. [ABSTRACT FROM AUTHOR]

The reaction of beef kidney rhodanese with selenosulfate was studied. The selenium-treated enzyme shows an absorption spectrum with a maximum at 375 nm attributable to a sulfoselenide group. This absorption is bleached by addition of cyanide. After cyanide treatment stoichiometric amount of selenocyanate can be found. The intrinsic fluorescence of rhodanese is quenched by addition of stoichiometdc selenosulfate. This effect can be reversed by cyanide or sulfite but not by selenite or glutathione. By comparison with model complexes the selenium-rhodanese intermediate was identified as a cysteinyl-selenium derivative. [ABSTRACT FROM AUTHOR]

The substrate specificity of pantethinase, the mammalian enzyme which has been isolated from horse kidney cortex, has been studied. The real substrate has been found to be pantetheine in the reduced form, and the involvement of one or more —SH groups of the enzyme is also considered. A number of other compounds, chemically related to pantetheine, has also been tested as substrates. Pantethinase is able to metabolise pantetheine-4′-phosphate, with an activity about 10% of that exhibited for pantetheine; it is inactive on the other substances tested (pantothenoyl- cysteine-4′-phosphate, pantothenoylcysteine, pantetheine thiazoline, CoA). The metabolic role of pantethinase seems to be, in the light of the above results, connected with a degradative pathway of pantethcine-4′-phosphate, which is known to be one of the intermediates in the biosynthesis of CoA. [ABSTRACT FROM AUTHOR]

Cysteamine oxygenase has been further purified to a degree where it appeared homogeneous in disc gel electrophoresis. The purified enzyme possesses a molecular weight of 52000 when it is treated with either 8 M urea or with 0.1 % sodium dodecyl sulfate, The molecular weight of the native undissociated enzyme was 96000 when determined by dextran gel filtration and 92000 when determined by sedimentation equilibrium in the ultracentritfuge. These results are consistent with the occurrence of two similar subunits in the molecule of cysteamine oxygenase. [ABSTRACT FROM AUTHOR]

The addition of sodium sulfide to disulfide-containing proteins disolved in 0.01 N NaOH produces an absorption in the range of 320-350 nm similar to that exhibited by cystine or eystamine treated under the same conditions. Proteins free of cystine and cysteine and proteins containing disulfide bonds which have been reduced with sodium borohydride do not produce this .absorbance. Urea and guanidine at 6 M concentration increase the rate of production of the absorbance while sodium dodecyl sulfate at concentration of 0.02 M has an inhibitory effect both on the rate and the extent of the absorbance. The effect of pH, concentration of reagents and of the additions of cyanide and hypotaurine has been studied. The 320-350 nm absorbance as been related to the production of persulfide groups (R-SSH) as a result of the cleavage of disulfide bonds by sulfide. Attempts to prepare a purified model protein containing persulfide groups have failed owing to the instability of the persulfide group in conditions far from those used for its preparation. [ABSTRACT FROM AUTHOR]

Cysteamine oxygenase has been analysed for the presence of metals, by spectrographic analysis, and X-ray fluorescence. Iron, copper, and zinc have been found in the enzyme in the quantity accounting for 1 atom of each metal per molecule of enzyme. Passage of the enzyme through a chelating resin column did not appreciable change the specific activity but the metal content was decreased below the value of one atom per molecule of enzyme. Possible explanations for this finding are suggested. The effect of several chelating agents on enzymic activity has been studied. All the chelating agents assayed were found inhibitory when tested in suitable concentration. The highest inhibition was observed with 8-hydroxyquinoline, diethyldithiocarbamate and o-phenanthroline. KCN also was a good inhibitor. It is concluded that cysteamine oxygenase is a metal-containing enzyme and that the metals should play a functional role. [ABSTRACT FROM AUTHOR]

1. Intrinsic fluorescences of the sulfur-transferring protein rhodanese (or thiosulfate : cyanide sulfurtransferase) and of artificially persulfurated bovine serum albumin were studied. Both are attributable mainly to tryptophan side-chains. Addition of cyanide or sulfite to these proteins causes a 20-25% increase in fluorescence. 2. Both proteins exhibit a low intensity absorption band at about 330 nm which disappears on addition of cyanide or sulfite. This absorption can be restored in rhodanese, after the treatment with cyanide, by excess thiosulfate; in the same time the fluorescence decreases to that of sulfur-containing enzyme. 3. A change in solvent viscosity, obtained by addition of glycerol, reduces the quenching effect in both proteins. On the basis of these data a long-range energy-transfer mechanism between the donor (tryptophan) and an acceptor is proposed. The acceptor involved seems to be one or more persulfide groups (R-SSH) in both proteins. 4. The presence of a persulfide group into rhodanese fits very well with the current knowledge of the mechanism of this enzyme. [ABSTRACT FROM AUTHOR]