Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation.

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase.

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.

Polymorphism of rat gastric mucosal histidine decarboxylase: effects of the coenzyme and sulfhydryl groups.

Several factors are examined for their implication in the charge heterogeneity and form conversion of rat gastric mucosal histidine decarboxylase. The apoenzyme and the holoenzyme are undistinguishable with respect to their pI and to the distribution of enzyme activity in the three forms. The latter are not produced by differential coenzyme binding. Studies for glycoprotein characterization provide evidence that the heterogeneity does not arise from enzyme-bound carbohydrate. Oxidative or reductive environments change the distribution between forms without modifying the molecular weight. Conversion of form III to forms I and II can be effected by treatment with dithiothreitol. A similar loss of negatively charged form occurs upon ageing and is not prevented by an alkylating agent. All three forms show equal sensitivity to N-ethylmaleimide and dithiothreitol inhibitions. The oxidation-reduction state of exposed sulfhydryl groups may be responsible at least in part for the charge heterogeneity.

Histidine decarboxylase from rat gastric mucosa: heterogeneity and enzyme forms modification.

Rat gastric mucosal histidine decarboxylase is shown to exist in the crude extract as three active charged forms which are separable by isoelectric focusing. The distribution of enzyme activity in the three forms is independent of the homogenizing medium and of the isoelectric focusing procedure indicating that the heterogeneity does not arise during isolation. Multiple forms correspond to histidine decarboxylase and are related neither to 3,4-dihydroxyphenylalanine decarboxylase nor to the result of aggregation. This charge difference between the enzyme forms changes according to the time of storage and to the temperature, leading to the generation of less negatively charged species. The conversion cannot be attributed to proteolytic degradation nor to differences in stability between forms. The data indicate that these alternative charged states may really result from an in vivo post-translational modification of the enzyme.

Diurnal variations of S-adenosyl-L-methionine and adenosine content in the rat pineal gland.

S-adenosylmethionine and adenosine levels in the rat pineal gland were determined by high-performance liquid chromatography after fractionation of the pineal extracts. The concentration of S-adenosylmethionine follows a circadian rhythm and is about three times higher during the day (2.5 nmol/gland) than the night (1.1 nmol/gland). The variations in the level of adenosine are apparently more complex. Over the 24 hours period there are two maxima at 03.00 (120 pmol/gland) and 15.00 hrs (100 pmol/gland) and one minimum at 09.00 hrs (50 pmol/gland). In addition, only an ultradian rhythm with a period of 12 hrs and an acrophase of 3 hrs can be evinced by computer analysis.

Specific determination of alpha-tocopherol in food and feed by fluorometry. Part 1. Manual method.

The proposed fluorometric method for determining alpha-tocopherol is highly specific and sensitive, yet requires low-cost equipment available in any laboratory. It is robust and fairly fast (4 determinations in 100 min, sample preparation not included). It has been tested in parallel with a conventional thin layer chromatographic method on foods and feeds. The only necessary cleanup is the usual saponification. The unsaponifiable fraction can be extracted with ethyl ether or, preferably, with Extrelut columns. Isooctane is used as a carrier solvent. Reagents and their solvents are added to the isooctane solution before each successive reaction and are then eliminated by partition with water. The alpha-tocopherol (alpha-T) derivative always remains in isooctane. The first step is nitrosation and elimination of tocopherols and tocotrienols other than alpha-isomers. alpha-T is then oxidized to alpha-tocored (alpha-TR) with a mixture of sulfuric acid, ferric chloride, and iodine bromide. alpha-TR is then condensed to a new reagent: 4,5-dimethyl-o-phenylenediamine. The phenazine formed is strongly fluorescent. Iodine and bromine add to the double bonds of alpha-tocotrienol present and quench the fluorescence of its phenazine. A procedure for blank assays specifically inhibits the conversion of alpha-T to alpha-TR.

Automated determination of alpha-tocopherol in food and feed. Part 2. Continuous flow technique.

The proposed determination of alpha-tocopherol is a continuous flow method with fluorometric detection. The only cleanup necessary is the usual saponification. A solution of the unsaponifiable matter in isooctane is automatically assayed. Isooctane is the carrier solvent and extractions are inserted between steps. These steps are selective reactions which render the method very specific. The natural homologs of alpha-tocopherol (alpha-T) do not interfere in the determination. A procedure for blank assays allows selective inhibition of alpha-T conversions and measurement of interfering fluorescence. The method is highly sensitive, which allows the determination of alpha-T in very dilute solutions. This in turn suppresses matrix effects and renders the results reliable. The time interval between 2 peaks is 6 min, washing included, and it is possible to carry out 50 determinations per day (sample preparation not included). The system is robust and maintenance is easy. Parallel determinations of foods and feeds have been carried out with a conventional thin layer chromatographic method.

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.

Properties of histidine decarboxylase from rat gastric mucosa.

The properties of specific histidine decarboxylase from highly purified rat gastric mucosa preparations were studied. The kinetic parameters were pH dependent: the apparent Km value varied inversely with pH; the maximum reaction velocity was reached at pH 6.6; the optimum pH was related to substrate concentration. The enzyme was unstable below pH 5.5. The effect of temperature was investigated and the enzyme activity was optimum near 56 degrees C. The thermal inactivation of the enzyme showed the presence of several active forms displaying distinct thermostabilities. The effect of coenzyme and substrate on heat stability was established. A small amount of pyridoxal phosphate was required for maximum enzyme activity, and the Km was low. The cofactor appeared to be tightly bound to the apoenzyme; nevertheless there was a fraction more easily resolved by dialysis. With high pyridoxal phosphate concentrations non-competitive inhibition occurred. Histamine inhibited the enzyme at high concentrations, the inhibition being competitive with respect to the substrate. No metal ion was required for enzyme activity; the enzyme was inhibited by sulfhydryl reagents and heavy metal ions, and also by high concentrations of reducing agents. The tryptophan residue of the holoenzyme seemed to be essential for the catalytic process.

The regional concentrations of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, and adenosine in rat brain.

The concentrations of S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 65 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (-25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100--180%.

S-adenosyl-L-homocysteine: simplified enzymatical preparation with high yield.

S-adenosyl-L-homocysteine hydrolase catalyses the synthesis of S-adenosyl-homocysteine (AdoHcy) from adenosine (Ado) and L-homocysteine (L-Hcy) in the absence of other enzymes, such adenosine deaminase, using Ado or L-Hcy as a substrate. Studies concerning the effect of substrates and enzyme concentrations with enzyme from beef liver, an inexpensive enzyme source, allowed the determination of the smallest amount of enzyme leading to total conversion to AdoHcy for the highest Ado and L-Hcy concentrations. Washing out the blood, homogenization of beef liver slices at pH 4.9 and precipitation with between 40% and 50% saturated (NH4)2SO4 eliminated contamination of tissue by blood adenosine deaminase. This enzyme preparation transforms Ado only into AdoHcy in the presence of L-Hcy. In a 1 1 final volume, an amount of enzyme preparation corresponding to at least 18.5 mg of fresh liver causes the total transformation of 60 mM Ado in the presence of 90mM L-Hcy after 40 hours incubation at 37 degrees C and pH 6.8. After purification by two crystallizations, the AdoHcy yield, based upon the Ado incubated, is about 80% of theory (1.0 g of AdoHcy per g of fresh beef liver).

[S-adenosyl-L-homocysteine: 2. An anticonvulsant]. / La S-adénosyl-L-homocystéine: 2. Anticonvulsivante.

S-Adenosyl-L-homocysteine (SAH) decreases the epileptiform discharges induced by hippocampic stimulation. SAH reduces the pentetrazol convulsions, does not modify picrotoxine convulsions, and increases strychnine toxicity. These effects are not linked to gamma-aminobutyric acid metabolism perturbation or to the concentration of some amino acids known for their action in synaptic transmission.

[S-adenosyl-L-homocysteine: 1. Influence on sleep]. / La S-adénosyl-L-homocystéine: 1. Inductrice de sommeil.

S-Adenosylhomocysteine, 0.1-20 mg/kg, influences the sleep patterns of rat, cat and rabbit by increasing the slow-wave and fast-wave sleep for 6 h. The SAH effects are increased by p-chlorophenylalanine and iproniazid and unchanged by reserpine. SAH effects are correlated with modification of norepinephrine and serotonin metabolism.

[Identification of 4-O-methyldopamine in rat tissues by reverded-phase liquid chromatography (author's transl)]. / Identification de la 4-O-méthyl dopamine dans les tissus de rat par chromatographie liquide en phase inverse.

4-O-Methyldopamine was identified and assayed in tissues from L-dopa treated rats by reversed-phase high-performance liquid chromatography. The initial steps in the separation of catecholamines were performed by alumine, a weak cation-exchange resin, and thin-layer chromatographic techniques. After L-[3 H] dopa administration, the radiochromatogram was superimposed on the fluorochromatogram obtained with authentic marker 4-O-methyldopamine. This metabolite was detected in kidney but not in brain. The 4-O-methyldopamine:3-O-methyldopamine ratio was 0.032 in kidney. The influence of various treatments on this ratio was investigated. A 160% increase was found after L-dopa administration. This effect was potentiated by nialamide pretreatment (550% increase).

[Rat liver S-adenosyl-L-homocysteine hydrolase purification by affinity column chromatography (author's transl)]. / Purification de la S-adénosyl-L-homocystéine hydrolase du foie de rat par chromatographie d'affinité.

S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been purified 240-fold from rat liver by affinity column chromatography on aminohexyl sepharose bound 6-mercaptopurine 9 D-riboside. The purified enzyme was homogeneous by gel electrophoresis.

Activation by S-adenosylhomocysteine of norepinephrine and serotonin in vitro uptake in synaptosomal preparations from rat brain.

S-Adenosylhomocysteine (10(-7)-10(-5) M) activated norepinephrine (NE) and serotonin (5HT) in vitro uptake in synaptosomal preparations from rat brain, but did not affect dopamine (DA) uptake. When administered to rats (7 mg/kg i.p.), it has the same effect on in vitro NE and 5HT uptake. It did not affect NE and 5HT release.

Action of S-adenosyl-L-homocysteine on the metabolism of dopamine, norepinephrine and serotonin in rat brain.

S-Adenosylhomocysteine (SAH) (0.1 to 30 mg/kg/i.p.) increased 3H-norepinephrine synthesis from 3H-tyrosine in the rat brain stem and mid brain, but did not alter its tissular level. It decreased both the serotonin level and the formation of 3H-serotonin from 3H-tryptophan and increased the formation of 3H-5hydroxyindolacetic acid. Its effects began 1 hr after administration and was still present 6 hr later. It did not alter dopamine metabolism. A correlation between the hynotic effect of SAH and the modification of the metabolism of cerebral monoamines was pointed out.

[Effect of S-adenosyl-L-homocysteine of dopamine catabolism]. / Effet de la S-adenosyl-L-homocysteine sur le catabolisme de la dopamine.

1.--The administration of SAH to rats, at physiologically active dose on the sleep, does not change the urinary level of MD and NM. On the other hand, the excretion of DA and NA decreases. 2.--In the brain, SAH does not modify neither the concentration of NA and NM in hypothalamus and thalamus, nor the concentration of DA and MD in corpus striatum. 3.--After intracisternally injection of [14C]DA or [3H]NA, SAH increases the level of [14C]MD and [3H]NM. 4.--Contrary to the studies in vitro, where SAH is an inhibitor of COMT, on the rat it does not seem prevent the methylation of DA and NA.

[Effects of S-adenosylmethionine and S-adenosylhomocysteine on in vivo synthesis of noradrenaline and serotonin in different parts of the rat brain]. / Effets de la S-adénosylméthionine et de la s-adénosylhomocystéine sur la synthése, in vivo, de la noradrénaline et de la sérotonine dans différentes parties du cerveau de rat.

S-adenosylmethionine (0,1 mg/kg/ip) decreases in vivo, norepinephrine (NE) synthesis and does not affect 5-hydroxytryptamine (5 HT) synthesis in the Rat brain. S-adenosylhomocysteine (7 mg/kg/ip) increases NE synthesis and decreases 5 HT synthesis. Neither nucleoside affects dopamine synthesis.