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PubMedDate not supplied

The stability of 5-aminolevulinic acid and its ester derivatives.

Application of 5-aminolevulinic acid (ALA) and its esters for treatment of cancer and other disorders became a rapidly developing branch of photodynamic diagnosis and therapy. For clinical use, solutions of ALA (must be buffered to physiological pH. Unfortunately, in such conditions they are unstable. Two molecules of ALA condense and 2.5-(beta-carboxyethyl)-dihydropyrazine and 2.5-(beta-carboxyethyl)-pyrazine are formed. Although, numerous eleborations relate to stability of ALA alone very little or none is known about the stability of ester derivatives of ALA. The present investigations have comprised the stability of ALA and its esters at 37 degrees C in respect of pH. concentration and time of reaction. The studies showed that ALA esters alike ALA undergo dimerisation at pH near 5. Moreover, it is possible that at pH>5.5 hydrolysis of the esters occurs, but this elaboration do not comprise explanation of this phenomenon.

Aminolevulinic Acid↗

PubMed2004

Analysis of roasted coffee bean volatiles by using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

The volatile headspace from two coffee bean varieties, namely Arabica (Coffea arabica) and Robusta (Coffea canephora ex Froehner), were sampled by using solid-phase microextraction (SPME), and then analysed with comprehensive two-dimensional gas chromatography interfaced to a time-of-flight mass spectrometer (GC x GC-TOFMS). Two distinct column set combinations were investigated--an apolar-polar and polar-apolar configuration--and the separation achieved from each set was compared. Results were compared with a previous literature report for coffee analysed by GC x GC-FID, using an analogous polar-apolar column set combination, where authentic standards were used to confirm the position of the selected components in the 2D separation space. The present study provides independent mass spectral confirmation of component identity, and demonstrates that the relative, structured position of these components is comparable in the two experiments. Total ion current (TIC) chromatograms were processed using ChromaTOF automated data processing software. It was necessary to restrict the number of processed peaks to 1000 (S/N > 100), which required approximately 8 h for processing. Extracted ion chromatograms were generated using prominent fragment ions, and unique masses, to aid in analyte identification process, and was particularly useful in instances of component peak overlap, and for the identification of pyrazine analytes (e.g. 44, 88, 122 u). Semi-quantitative analysis was restricted to the 44 selcted components; however, the omission of peaks with S/N < 100, limiting the processed peaks to 1000, reduced the semi-quantitative application of the GC x GC-TOFMS method developed. Finally, results gained from GC x GC-TOFMS and GC x GC-qMS analyses were comparable with respect to spectral similarity assignments for the 44 target analytes.

Coffee↗

PubMedDate not supplied

Association of enhanced superoxide generation by neutrophils with low superoxide scavenging activity of the peripheral blood, joint fluid, and their leukocyte components in rheumatoid arthritis: effects of slow-acting anti-rheumatic drugs and disease activity.

To study the role of the superoxide radical (O2-) in the pathogenesis of rheumatoid arthritis (RA), both the O2- generation of peripheral blood (PB) polymorphonuclear cells (PMN) and the superoxide scavenging activity (SSA) of PB-PMN, plasma, joint fluid (JF) and PB- or JF-mononuclear cells (MNC) were measured in forty-five patients with RA using the highly sensitive and specific 2-methyl-6-methoxyphenyl-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one dependent chemiluminescence and the electron paramagnetic resonance/spin trapping methods, respectively. Since many drugs, particularly the slow-acting anti-rheumatic drugs (SARDs) used in RA, may alter O2- metabolism, the effects of SARDs on SSA were also studied. The basal O2- release and opsonized zymosan- or phorbol myristate acetate-stimulated O2- generation by PB-PMN from RA patients were significantly increased, while the SSA of PMN was decreased as compared to those from healthy controls. In addition, the SSA of PMN showed a negative correlation with their O2- generation rates. The JF-PMN showed lower SSA levels than PB-PMN. A negative correlation was also found between the SSA of the plasma and the erythrocyte sedimentation rates. The SSA of the plasma, PB-PMN, JF and JF-PMN were significantly higher in patients treated with SARDs than those without. In cell-free systems, sulfasalazine (SAP) and its metabolite, 5-amino-salicylic acid, had a direct SSA within a millimolar range. The other metabolites of SASP and D-penicillamine had an indirect SSA, since they affected the O2- generating system. Auranofin and bucillamine had no SSA. However, none of the SARDs examined could scavenge O2- at concentrations reported in patients' plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

5-hydroxytryptamine (5-HT)1A receptors and the tail-flick response. I. 8-hydroxy-2-(di-n-propylamino) tetralin HBr-induced spontaneous tail-flicks in the rat as an in vivo model of 5-HT1A receptor-mediated activity.

This study pharmacologically characterizes a novel behavioral response as a potential in vivo model of serotonin (5-HT)1A receptor-mediated activity. In rats restrained in horizontal cylinders, the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin HBr (8-OH-DPAT), dose-dependently (0.04-10.0 mg/kg s.c.) elicited spontaneous tail-flicks (STFs). This action was mimicked by other ligands possessing high affinity and high efficacy at 5-HT1A sites: RU 24969 [(5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole], lisuride, (+)-lysergic acid diethylamide and 5-methoxy-N,N-dimethyltryptamine hydrogen oxalate. The response could not be elicited by CGS 12066B [7-trifluormethyl-4-(4-methyl-l-piperazonyl)-pyrrolol- [1-2-a] quinoxaline dimaleate], mCPP 1-(3-chlorophenyl)-piperazine-2-HCl, TFMPPm-trifluromethylphenylpiperazine HCl, MK 212 [6-chloro-2-(l-piperzinyl)pyrazine], quipazine and DOI (+-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane HCl, which act in vivo as agonists at 5-HT1B, 5-HT1C and/or 5-HT2 receptors, or by the 5-HT3 agonist, 2-methyl-5-HT. p-chloroamphetamine, which releases endogenous 5-HT, also evoked STFs; in contrast, d-amphetamine, a preferential releaser of catecholamines, was inactive, as were agonists and antagonists at alpha-1, alpha-2, beta-1, beta-2, dopamine D1 and D2 sites. 8-OH-DPAT-elicited STFs were blocked by the 5-HT1/2 antagonist, methiothepin, but not by the 5-HT1C/5-HT2 antagonists, mianserin, ritanserin and ICI 169,369 [2-(2-dimethylaminoetheylthio)-3-phenylquinoline] nor by the 5-HT3 antagonists, GR 38032F [(1,2,3,9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-l-yl)methyl]-4H- carbazol-4-one HCl], ICS 205,930 [(3 alpha-tropanyl)-1H-indol-3-carboxylic acid ester] and MDL 72222 [(1 alpha H, 3 alpha, 5 alpha H)-tripan-3-yl-3,5- dichlorobenzoate]. beta-Blockers with 5-HT1A affinity i.e., (-)-alprenolol, (+/-)-isamoltane and, stereoselectivity, (-)-but not (+)-pindolol, blocked the action of 8-OH-DPAT. Spiperone and spiroxatrine, D2 antagonists with high 5-HT1A affinity, also inhibited 8-OH-DPAT-induced STFs. Selective beta-blockers and D2 antagonists with low 5-HT1A affinity were inactive. 5-HT1A partial agonists, the pyrimidinylpiperazines, buspirone, gepirone and ipsapirone, the halogenated phenylpiperazine, LY 165,163 [1-(2-(4-aminophenyl) ethyl-4-(3-trifluoromethylphenyl)-piperazine], and the benzodioxane, MDL 72832 [8-(4-(1,4-benzodioxan-2-yl-methylamino)-butyl-8-azaspiro-(4 ,5)-decane- 7,9-dione] did not elicit STFs and antagonized the effect of 8-OH-DPAT.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1990

Inhibitory effects of azelastine on superoxide anion generation from activated inflammatory cells measured by a simple chemiluminescence method.

Blood was drawn from healthy human volunteers and neutrophils and eosinophils were purified on a Conray-Ficoll and a Percoll gradient, respectively. Rat mast cells were also purified on a Percoll gradient. Superoxide anion (O2-) generation from the cells were measured by 2-methyl-6-[p-methoxy-phenyl]-3,7-dihydroimidazo[1,2-a]pyrazin+ ++-3-one (MCLA)-dependent luminescence. Addition of 0.5 mumol/l MCLA and a stimulatory agent, such as phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and compound 48/80, to a suspension of each cell caused a marked luminescence which was inhibited by 0.5 mumol/l superoxide dismutase (SOD). Azelastine (A-5610) significantly inhibited the O2- generation from each activated inflammatory cell in a dose-dependent manner. When eosinophils were activated by fMLP in the presence of cytochalasine (CB), azelastine abolished the luminescence stronger than that from the fMLP-stimulated cells in the absence of CB.

Animals↗

PubMedDate not supplied

[Achievements in the search for and creation of Soviet anthelmintic agents].

Basic achievements in the creation of antinematodal, anticestodal and antitrematodal agents are reviewed chronologically. It is shown that the search was performed mainly among the derivatives of benzimidazole, cyclic amidines, diphenylmethane, salicyl anilides, pyrazine isoquinolines, among natural products of avermictines and milbemicines. It is noted that nowadays there is every possibility for the treatment of mass helminthiases in the USSR. However, it has been found that some drugs are associated with a certain risk and therefore the necessity for further search of safe anthelminthic agents has been emphasized.

Anthelmintics↗

PubMed1988

Mechanisms of effects of intrathecal serotonin on nociception and blood pressure in rats.

The effects of intrathecal (i.t.) serotonin (5-HT) and a number of serotonergic receptor agonists on nociception and blood pressure were examined in rats. Intrathecal 5-HT produced dose-dependent inhibition of the nociceptive tail-flick reflex (ED50 = 100.0 micrograms) and dose-dependent depressor effects. The 5-HT1A agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin and the 5HT1B agonist 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU-24969) also produced depressor effects but, in contrast to 5-HT, facilitated the tail-flick reflex, whereas the 5-HT2 agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 6-chloro-2-(1-piperazinyl)-pyrazine (MK-212) and quipazine produced dose-dependent antinociception and had little or no effect on blood pressure. These results suggest that the antinociceptive and depressor effects of i.t. 5-HT are mediated by spinal 5-HT2 and 5-HT1 receptors, respectively. In other experiments, rats chronically treated with i.t. 5-HT developed tolerance to its antinociceptive effects, whereas chronic i.t. morphine or clonidine did not produce cross-tolerance to i.t. 5-HT. These results suggest that serotonergic spinal antinociceptive mechanisms are distinct from the mechanisms by which opioid receptor and alpha-2 adrenoceptor agonists produce antinociception in the spinal cord.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1989

Prolactin-lowering ability of (+/-)-idazoxan may be linked to a central noradrenergic-serotonergic interplay.

2-[2-(1,4-benzodioxanyl)-2-imidazoline] (idazoxan) sometimes lowers basal prolactin levels in the male adult rat, but strongly inhibits hyperprolactinemia in suckling rats. A possible antido paminergic drug effect is not involved, due to its inability to modify prolactin release from superfused pituitary in vitro as well as rat haloperidol hyperprolactinemia in vivo. On the contrary, in the rat idazoxan counteracts hyperprolactinemias due to central presynaptic serotonergic neurotransmission increase (5-hydroxytryptophan, D-fenfluramine and fluoxetine) but not those related to direct agonists at 5-hydroxytryptamine (5-HT) receptor (6-chloro-2-[1-piperazinyl] pyrazine, MK 212; 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane, DOI). (+/-)-Idazoxan does not modify [3H]-5-HT release from isolated hypothalamic synaptosomes. It displays an affinity for alpha-2 adrenergic autoreceptors about 250-fold more than for alpha-2 heteroreceptors located on 5-HT nerve terminals in the central nervous system (pA2 value 9.99, equivalent to a Kb of 0.1 nM vs. pA2 7.60 equivalent to a Kb of 2.5 nM). The noradrenergic outflow selectively induced by idazoxan in the brain may negatively modulate the 5-HT release from the relevant nerve endings, thus preventing prolactin release due to an activation of presynaptic serotonergic axons induced both physiologically (lactation) and pharmacologically (5-hydroxytryptophan, D-fenfluramine and fluoxetine) without influencing hyperprolactinemias related to a direct activation of serotonergic receptors (MK 212 and DOI). Other blocking agents, strong but less selective than (+/-)-idazoxan for noradrenergic brain neurotransmission, do not modify or increase blood prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1989

Inhibition of monoamine oxidase by analogues of amiloride.

Amiloride analogues with nonaromatic substituents on the 5-amino group or different substituents on carbon-6 of the pyrazine ring were tested as inhibitors of monoamine oxidase A and B in rat brain homogenate. The inhibition was competitive and reversible. 5-(N,N-Tetramethylene)amiloride protected the A type in the homogenate against irreversible inhibition by clorgyline. A reciprocal relation was found to exist between inhibitory constants of 5-N-substituted amiloride analogues for monoamine oxidase A and the ratio of overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from the isolated rat tail artery incubated in the presence of a 50 microM concentration of the analogue, when the tissue was exposed to 10 microM tyramine. The 5-amino group appeared to be essential for inhibition of the A but not of the B type. Bell-shaped relations between inhibitory constants of 5-(N-alkyl)- and 5-(N,N-dialkyl)-substituted analogues and lengths of alkyl chains were different for each type. The presence of a methyl group in the alpha-position of the chain increased substantially the inhibitory constant for the A type. Halogen atoms as substituents on carbon-6 increased inhibitory constants for both types of the enzyme in the sequence: I less than Br less than Cl less than F. These findings are consistent with the existence of hydrophobic binding sites of restricted dimensions in both types of the enzyme.

Adrenergic Fibers↗

PubMed1986

Neurochemical approaches to the prevention of ventricular fibrillation.

Current evidence indicates that susceptibility to ventricular fibrillation (VF) can be reduced by decreasing cardiac sympathetic activity and by increasing vagal tone. Pharmacological agents that favor such a pattern of autonomic outflow protect the heart against fibrillation. These include morphine sulfate, clonidine, digitalis drugs, and bromocriptine. An intriguing new approach involves changing the serum concentration of amino acid precursors of the central neurotransmitters that modulate autonomic traffic. Considerable evidence indicates that accumulation of serotonin in the brain reduces sympathetic neural activity. When L-tryptophan or 5-hydroxytryptophan is administered with phenelzine (a monoamine oxidase inhibitor) and carbidopa (a selective peripheral L-amino acid decarboxylase inhibitor) to raise brain serotonin, a significant increase in myocardial electrical stability is observed. This effect results from a decrease in cardiac sympathetic tone as indicated by selective denervation and nerve recording studies. Enhancing serotoninergic neurotransmission can also significantly reduce vulnerability to VF during acute coronary artery occlusion. The effect of augmenting serotoninergic activity without the use of enzyme inhibitors has been investigated by administering agents such as melatonin, 5-methoxytryptophol, and 6-chloro-2-(1-piperazinyl) pyrazine (MK-212). Each of these substances increases cardiac electrical stability. The protective influence is unaffected by bilateral vagotomy but is blocked by the specific serotonin antagonist methergoline. Diminution of cardiac sympathetic drive appears to be the main mechanism of action. Thus, neurochemical interventions can exert a profound effect on cardiac electrical stability. Recent advances in neurochemistry and psychopharmacology promise new insights into the problem of sudden death and suggest a fresh approach for the management of life-threatening arrhythmias.

Animals↗

PubMed1987

Relative efficacies of piperazines at the phosphoinositide hydrolysis-linked serotonergic (5-HT-2 and 5-HT-1c) receptors.

Serotonin (5-HT)-stimulated phosphoinositide hydrolysis is mediated by the 5-HT-2 receptor in rat cerebral cortex and by the 5-HT-1c receptor in rat choroid plexus. These systems were used to determine relative efficacies of piperazine derivatives at the 5-HT-2 and 5-HT-1c receptors. Both quipazine and 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) stimulated phosphoinositide hydrolysis in cerebral cortex, and these effects were blocked by ketanserin. The maximum responses to these agonists were 80% of the maximum response to 5-HT. m-Trifluoromethylphenylpiperazine (TFMPP), m-chlorophenylpiperazine (MCPP) and 1-(1-naphthyl)-piperazine (1-NP) did not stimulate phosphoinositide hydrolysis in cerebral cortex at concentrations that blocked the effect of 5-HT. In the choroid plexus, TFMPP and MCPP, as well as MK-212 and quipazine, increased phosphoinositide hydrolysis and mianserin blocked these effects. MK-212 had an efficacy which was equal to that of 5-HT, whereas quipazine, MCPP and TFMPP were partial agonists in the choroid plexus. 1-NP did not stimulate phosphoinositide hydrolysis in choroid plexus but completely blocked the effect 5-HT. On the basis of these data, we conclude that quipazine and MK-212 are partial agonists at 5-HT-2 receptors in cerebral cortex, whereas 1-NP, TFMPP and MCPP are pure antagonists of the cortical 5-HT-2 receptor. However, TFMPP and MCPP as well as quipazine and MK-212 are agonists at the 5-HT-1c receptor, while 1-NP is a pure antagonist of the 5-HT-1c receptor in choroid plexus.

Animals↗

PubMed1987

Pharmacodynamic effects of the sleep inducer zopiclone.

Pharmacodynamic effects of [6-(5-chloro-2-pyridyl)-6,7-dihydro-7-oxo-5H-pyrrolo[3,4-b]pyrazin -5- yl]-4-methyl-1-piperazine-carboxylate (zopiclone, RP-27267), chemically unrelated to benzodiazepines and a potential new sleep inducer, on the peripheral system were investigated in several species of animals. The drug was dissolved in the vehicle of 0.01 mol/l HCl solution for intravenous administration or for addition to the bath medium and was suspended in 0.25% carboxymethylcellulose solution for oral administration. In unanesthetized rabbits, zopiclone, 0.5 mg/kg i.v., exerted no action and at 1 mg/kg slightly decreased respiration and heart rate without affecting blood pressure and ECG. Zopiclone at 10(-6) g/ml had no action in the isolated guinea-pig atria but at 10(-5) g/ml it produced a gradual and slight decrease in heart rate without affecting the contraction. In the isolated small intestine of rabbits and guinea-pigs, zopiclone at 10(-6) g/ml had no action but produced a slight inhibition in a dose of 10(-5) g/ml. Zopiclone, 10(-5) g/ml, did not affect the stimulatory effects of acetylcholine, serotonin, histamine and barium in the isolated guinea-pig intestine. Zopiclone, 1, 5 and 10 mg/kg i.v., exerted no action on rabbit intestinal movement in vivo. Zopiclone, 5, 10, 20 and 50 mg/kg p.o., had no effect on the propulsive motility of the mouse intestine. Zopiclone, 10(-5) g/ml, did not affect contractile movement of the uterus isolated from rabbits and did not influence the contractile response to epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

The odorant-sensitive adenylate cyclase of olfactory receptor cells. Differential stimulation by distinct classes of odorants.

We have characterized odorant-stimulated adenylate cyclase activity in isolated chemosensory cilia prepared from frog and rat olfactory epithelium. Cilia from both species exhibit high levels of adenylate cyclase activity. Basal activity is stimulated approximately 2-fold by GTP and approximately 5-fold by guanosine 5'-(3-O-thio)triphosphate and forskolin. Odorants augment enzyme activity 30-65% above the basal level in a tissue-specific and GTP-dependent manner. Calcium reduces GTP-stimulated activity with a 50% effective concentration at 10 microM. Odorants vary in their influence upon olfactory adenylate cyclase activity. Most fruity, floral, minty, and herbaceous odorants stimulate the enzyme. 3,7-Dimethyl-2,6-octadienenitrile (citralva), menthone, D-carvone, L-carvone, and 2-isobutyl-3-methoxypyrazine display similar potencies in activating the adenylate cyclase upto concentrations of 100 microM. Putrid odorants, such as isovaleric acid, triethylamine, pyridine, thiazole, and methoxypyrazine, and odorous chemical solvents, do not stimulate enzyme activity. In homologous series of pyrazine, thiazole, and pyridine odorants, compounds with the longest hydrocarbon side chains are best able to enhance enzyme activity. The failure of certain odorants to affect adenylate cyclase activity suggests that additional transduction mechanisms besides the formation of cAMP are involved in olfaction.

Adenylyl Cyclases↗

PubMed1979

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.

Binding Sites↗

PubMed1985

An experimental study of the antileprosy activity of a series of thioamides in the mouse.

A series of substituted thioamides have been studied to establish whether their structure-activity pattern against Mycobacterium leprae is similar to that displayed against M. tuberculosis. Antileprosy activity was evaluated in the mouse foot pad using both the kinetic and continuous methods. Ethionamide and prothionamide were found to be the most active compounds and to be of approximately equal potency. Thioisonicotinamide was about five times less active. 2-t-Butyl-thioisonicotinamide, 2-dimethylamino-thioisonicotinamide, and pyrazine carbonic thioamide were inactive at the dosages tested. High-pressure liquid chromatographic methods were devised to study the potential influence of pharmacological factors on their in vivo activity. Fecal measurements suggested that all of the thioamides were well absorbed when fed in the diet. After intravenous administration, all of the thioamides were rapidly eliminated from the mouse. The differences in their elimination rates probably played only a minor role in affecting their relative antileprosy activities. It was concluded that the structural requirements for antileprosy and antituberculosis activity of the thioamides are probably similar.

Amides↗

PubMed1986

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates.

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.

Candida↗

PubMed1985

Studies on the 4-carbon compound needed for the formation of the O-xylene ring of riboflavin.

The 4-carbon compound necessary for the formation of the o-xylene ring of riboflavin, was examined using labeled glucose and diacetyl in resting cells of Eremothecium ashbyii. The specific activity-time curves of riboflavin and lumichrome in the tracer experiments with [U-14C] glucose indicated that the specific activity of riboflavin during the incubation is much higher than that of its photolytic product, lumichrome. It is far above the level expected if the ribityl moiety of an intermediate, 4-ribitylamino-5-amino-2,6-dihydroxy-pyrimidine (RAADP), was to be utilized as the source of the 4-carbon unit on the o-xylene ring of riboflavin. It was further demonstrated that the specific activities of diacetyl and lumichrome at 10.5 h of the incubation and those of crystalline riboflavin and lumichrome at the later stage (20 h) of the incubation were identical respectively. Radioactive diacetyl furthermore proved to be likewise incorporated with a two fold dilution into riboflavin and lumichrome at 17 h of the incubation. The results reveal that it is not the ribityl fragment of RAADP bu the diacetyl that is the 4-carbon unit for the formation of the pyrazine ring of 6,7-dimethyl-8-ribityllumazine, that is, the o-xylene ring of riboflavin in the mold of Eremothecium ashbyii, although we still do not know whether the diketone requiring reaction is enzymatic or non-enzymatic.

Acetoin↗

PubMed1984

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. I. Characterization of Na+/Ca2+ exchange activity.

GH3 rat anterior pituitary cells possess a Na+/Ca2+ exchange transport mechanism which is present in purified plasma membrane vesicles prepared from these cells. Imposition of an outwardly directed Na+ gradient in vesicles results in a marked concentrative uptake of Ca2+ which is abolished by the Ca2+ ionophore A23187. Transport activity depends on a sustained Na+ gradient. Dissipation of the driving force by treatment with Na+ ionophores or by passive gradient collapse abolished transport activity. The exchange reaction is completely reversible since addition of extravesicular Na+ enhances Ca2+ efflux from Ca2+ loaded vesicles. A kinetic analysis of Na+/Ca2+ exchange indicates saturation kinetics for both substrates with apparent values of Km for Na+ and Ca2+ of 17 mM and 5 microM, respectively, and a Vmax of about 8 nmol/min/mg of protein for Ca2+ uptake at 25 degrees C. In addition to Na+/Ca2+ exchange, the transporter functions in a Ca2+/Ca2+ exchange mode with an apparent Km of 20 microM and Vmax of 16 nmol/min/mg of protein for Ca2+ influx. Na+/Ca2+ exchange is not inhibited by protonophores indicating that Ca2+ flux does not occur via coupled Na+/H+, Ca2+/H+ exchange. Transport is inhibited by derivatives of the pyrazine diuretic amiloride. The pH dependency of Ca2+ uptake displays a sigmoidal relationship with stimulation of activity at alkaline pH and inhibition at acid pH. Furthermore, the reaction is electrogenic (i.e. more than 2 Na+ transported per Ca2+) as demonstrated by stimulated uptake of lipophilic cations during exchange and by effects of artificially imposed membrane potentials on the rate of Ca2+ transport. Plasma membrane vesicles prepared from bovine anterior pituitary glands also display Na+/Ca2+ exchange with many of the same characteristics. These results support the notion that Na+/Ca2+ exchange functions in Ca2+ homeostasis in pituitary cells.

Amiloride↗