Increase of Brain Tryptophan caused by Drugs which stimulate Serotonin Synthesis

THE concentrations of tryptophan normally present in the mammalian brain are below the Michaelis constant (K_m) of tryptophan hydroxylase^1,2, suggesting that the rate of serotonin synthesis depends more on the concentration of brain tryptophan than on the amount of enzyme. We wish to report that various treatments which have been shown to increase brain serotonin synthesis also increase the concentration of tryptophan in brain. Conversely, p-chlorophenylalanine (PCPA), which inhibits serotonin synthesis³, decreases tryptophan in brain.     

Effect of <Emphasis Type="Italic">p</Emphasis>-Chlorophenylalanine on Tryptophan Hydroxylase in Rat Pineal

KOE and Weissman have demonstrated that p-chlorophenylalanine (pCPA) depletes serotonin in the brain of mammals¹. pCPA induces a variety of behaviour changes in rat, cat and other animals^2,3, presumably because of the depletion of serotonin in the brain. The biochemical mechanism of the depletion remains, however, to be elucidated, although it has been proposed that pCPA might inactivate tryptophan hydroxylase in the brain⁴. We demonstrate here that pCPA does not inactivate tryptophan hydroxylase in rat pineal, although pCPA depletes the serotonin level in the pineal.     

Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K_m relation to the substrate tryptophan (690 µM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K_m values with corresponding substrates. This suggests that the biosynthesis of serotonin or serotonin-derived secondary metabolites is restricted to cellular stages when high tryptophan levels are present.Key words: feruloylserotonin, serotonin, tryptamine, tryptamine 5-hydroxylase, tryptophan, tryptophan biosynthesis, tryptophan decarboxylaseSerotonin is found in a broad range of plants and is abundant in reproductive organs, such as fruits and seeds.^1–3 Even though many physiological roles for serotonin in plants have been proposed,^2–7 its actual roles have yet to be examined in detail using molecular, biochemical and genetic approaches. In plants, serotonin is synthesized by two enzymes: tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). TDC decarboxylates tryptophan into tryptamine, after which T5H hydroxylates tryptamine into serotonin.^8–10 TDC expresses at an undetectable level in rice leaves, whereas T5H expresses constitutively.^11,12     

Methanol elicits the biosynthesis of 4-coumaroylserotonin and feruloylserotonin in rice seedlings

Rice (Oryza sativa cv. Dongjin) plants responded to treatment with methanol by inducing the synthesis of secondary metabolites such as serotonin derivatives, which include feruloylserotonin and 4-coumaroylserotonin. This response was not only a dose dependence on methanol showing a maximum effect with 1% methanol concentration, but also methanol specific. No other solvents such as ethanol, atetaldehyde, isopropanol, formaldehyde and formic acid showed the induced synthesis of serotonin derivatives as methanol did. The methanol induced synthesis of serotonin derivatives was completely blocked by the addition of abscisic acid (ABA), and significantly inhibited by the additions of zeatin and indoleacetic acid (IAA). However, gibberellic acid (GA) had little effect on the action of methanol. Finally, the induced synthesis of serotonin derivatives upon methanol treatment was closely associated with the transient increase in the activity of key enzyme of serotonin N-hydroxycinnamoyl transferase (SHT) which catalyzes the condensation of serotonin and phenolic-CoA into serotonin derivatives.Key words: elicitor, methanol, 4-coumaroylserotonin, feruloylserotonin, serotonin N-hydroxycinnamoyl transferase, rice seedlingsElicitor broadly refers to molecules and stimuli that either induce or control gene expression and metabolism.¹ To date, a series of elicitors have been reported and include various cell wall constituents of plant and microbe origins, avirulence gene products from microbes, and a lots of chemical and physical stimuli such as CuSO₄, CuCl₂, ozone and UV light.² Among chemical elicitors, CuSO₄ is well known to elicit the accumulation of sesquiterpene lubimin in fruit cavities of Datura stramonium.³ Aluminum chloride (AlCl₃) induces resveratrol synthesis in grapevine leaves.⁴ It is also reported that Arabidopsis induces camalexin synthesis in response to α-aminobutyric acid.⁵ Recently, it was found that rice leaves upon senescence produced methanol which then triggered the synthesis of tryptophan and serotonin, suggestive of a key role of methanol as an endogenous elicitor for both primary and secondary metabolites.⁶ Here, we further examined the role of methanol in rice leaves as an elicitor on the biosynthesis of serotonin derivatives such as 4-coumaroylserotonin (CS) and feruloylserotonin (FS) which show antifungal activity as well as antioxidant activity.^7,8     

Activation of Brain Succinate Dehydrogenase by Lithium

IN spite of the widespread use of lithium salts as therapeutic and prophylactic agents against manic-depressive psychosis^{1, 2}, little is known about the mechanism of lithium effects^{3, 4}. Li⁺ stimulates noradrenergic neurones³, increases the uptake of this amine by isolated rat brain synaptosomes^{5, 6}, increases serotonin uptake into platelets obtained from manic-depressive patients⁷ and increases the levels of serotonin and tryptophan in rat brain⁸. On the other hand, Lallier observed inhibitory action of LiCl on dehydrogenase activity in homogenates of Rana fusca⁹. We have investigated whether long term treatment with Li⁺ affects brain succinate dehydrogenase activity.     

The <Emphasis Type="Italic">C1473G</Emphasis> polymorphism in gene <Emphasis Type="Italic">tph2</Emphasis> is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). Linkage between the C1473G polymorphism in gene tph2 causing the replacement of Pro⁴⁴⁷ by Arg⁴⁴⁷ in TPH-2 molecule and enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F₂ hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetically defined variability of enzyme activity in the mouse brain.     

Elucidation of the Central Serotonin Metabolism Pathway in Rhesus Macaques (Macaca mulatta) with Self-injurious Behavior

Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.

In the United States, mental illness affects up to 20% of adults⁶ and 22% of children.³³ One consequence of several mental health conditions, especially those associated with intellectual disabilities, is self-injurious behavior (SIB).²⁰ SIB has been defined as “behavior which produces physical injury to the individual’s own body.”⁴⁸ SIB is repetitive and persists over time, commonly manifesting as “pulling (hair or nails), scratching, hitting, banging and biting”.²⁰ Like many psychopathologies, SIB is a heterogeneous phenomenon and likely has numerous etiologies, but dysfunction of central serotonin signaling has been implicated in a number of human studies.^{15,17,19,22,32,43,44,46,47}Several risk factors have been identified for the development of SIB in laboratory rhesus macaques, including individual housing^24,29,31,38 and separation from the dam at an early age.^{3,24,30,38,39} A 10 y study at the National Institutes of Health (NIH) Animal Center found that rhesus macaques that are surrogate-peer-reared can have self-biting behavior at as young as 2 mo of age, were less social than their peers, and had a significantly higher incidence of self-biting than did mother-reared and peer-reared macaques.³⁰ Additional risk factors for SIB were identified in a study of 362 rhesus macaques at the New England Regional Primate Research Center as nursery rearing, housing individually at an early age, prolonged individual housing time, and more frequent total blood draws.²⁹ A study at the California National Primate Research Center examined behavioral data from over 4,000 rhesus macaques and found that males were more likely than females to develop self-biting, and that prolonged outdoor housing decreased incidence of self-biting.¹⁶ A paucity of environmental enrichment may also be correlated with SIB wounding behavior in short-term singly housed cynomolgus macaques.⁵³Managing macaques with SIB can be challenging because the macaques may repeatedly wound themselves over time, tend to rewound healed or partly healed wounds, and tend to make new wounds near previous wounds.¹³ In our experience, repeated wounding can be a significant problem requiring sedation and suturing of the new, larger wound. The wounds or altered behaviors may preclude use of the macaque in the intended research study or may necessitate the withdrawal of the macaque from a study to receive medical interventions. In addition to rendering a macaque potentially ineligible for the intended study, a research facility must dedicate additional resources to provide for the welfare and care of macaques with SIB. These macaques may require individual housing, increasing housing needs. However, care should be taken when relocating rhesus macaques with SIB as they can have greater incidence of SIB for to 1 y after the move.⁷ In addition, research facilities must invest more time, personnel, and resources into the psychologic wellness plan, veterinary care and monitoring, and enrichment plans for macaques with SIB.Rhesus macaques with SIB have long provided as a translational model for human SIB.^13,35 We previously demonstrated that impaired central dopamine signaling predicted the severity of SIB later in life in rhesus macaques.¹⁴ To date, serotonin dysfunction has been implicated in nonhuman primate SIB via successful treatments targeting that pathway.^11,12,55 Previous attempts to assay central serotonin function in animals with SIB found no differences compared with controls, though notably these studies did not measure serotonin directly.^25,49-51 Nevertheless, the authors of these studies themselves have pointed out that serotonin dysfunction remains a likely contributor to the phenomenon of SIB in both humans and nonhuman primates, and that further exploration is warranted as new methods and techniques become available.Serotonin is produced centrally by the metabolism of tryptophan and afterward converted into melatonin. However, central tryptophan may alternatively be metabolized into kynurenine. In 1969, one group proposed that a disturbance in this tryptophan metabolism away from the production of serotonin and shunting toward the production of kynurenine may be a major etiological factor of depression, a disorder not directly linked to SIB but similarly associated with serotonin dysfunction.²⁶ Central kynurenine and its metabolites are also associated with inflammatory conditions within the brain,¹⁸ and the proinflammatory cytokine Interleukin-6 (IL6) induces the kynurenine pathway,⁴² suggesting that a shunting of metabolism from serotonin to kynurenine could result in central dysfunction either by relative depletion of serotonin, increased inflammation in the brain, or both. Indeed, recent evidence suggests that the still-unclear mechanism by which the opioid antagonist naltrexone reduces SIB in humans and macaques could be due to its centrally active antiinflammatory properties.^{8,21,27,40,52}We hypothesized that SIB in macaques is associated with a pathologic change in homeostatic brain tryptophan metabolism, similar to that originally proposed by others,²⁶ with the aim of exploring how alterations in serotonin and its associated metabolic pathways found in macaques with SIB compare with data published in humans with other neuropsychiatric diseases that are associated with disturbances in the tryptophan metabolism pathways. More specifically, we hypothesize that, compared with macaques with normal behavior, macaques with SIB would have lower cerebral spinal fluid (CSF) serotonin levels, higher IL6 concentration as a marker of CNS inflammation, and a higher CSF kynurenine concentration due to the shift of tryptophan metabolism from serotonin production to kynurenine production. We used mass spectrometry as a novel way to assay the metabolites in the serotonin and kynurenine system directly in nonhuman primates (NHPs), which is more difficult to achieve in humans because of the necessary sampling.     

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [¹⁵N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[¹⁵N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[¹⁵N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (<Emphasis Type="Italic">SERT</Emphasis>) and degradation (<Emphasis Type="Italic">MAO-A</Emphasis>) gene expression in cultured rat serotonergic neuronal cell lines

Background

Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D₃ (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D.

Results

Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system.

Conclusions

These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

     

OH Radicals in Radiation Sensitization

CELLS can be sensitized to ionizing radiation by chemical compounds and this offers opportunity for investigating the mechanisms intermediate between energy absorption and expression of biological effect. Concentration dependence seems to be an important factor for the effectiveness of a radiation sensitizer. The inactivation constant k for X-irradiated suspensions of Bacillus megaterium spores shows a peak at 3 × 10^?3 M diacetyl (CH₃COCOCH₃) concentration¹. Although the mechanism of this effect is unknown, certain free radical reactions have been suggested^2–4. The decrease of k with increasing sensitizer concentration, envisaged as a protective action, indicates a competitive reaction in which the species contributing to the lethality are removed.     

Autoradiographic Localization of <Superscript>3</Superscript>H-GABA in Rat Retina

γ-AMINOBUTYRIC acid (GABA) is present in all layers of vertebrate retinae^1–3: in the rabbit retina it seems to be most concentrated in the ganglion cell layers² while in the frog it is concentrated primarily in cell layers which are rich in amacrine cells¹. Recent autoradiographic studies of the distribution of ³H-GABA in rat brain slices after incubation in vitro suggest that the labelled amino-acid is selectively concentrated by certain neural elements^4,5. In a study of the distribution of ³H-GABA in rabbit retina after injection of the labelled amino-acid into the eye, Ehinger⁶ found that radioactivity was accumulated principally in the inner plexiform, inner nuclear and ganglion cell and nerve fibre layers. Labelling was also concentrated in some cells occupying the same position as amacrine cells and in some nerve cells of the ganglion cell layer.     

Activation of the Protein Synthetic Capacity of Rat Liver Cell Sap by Amino-acids

CHANGES in the availability of amino-acids have marked effects on the rate of protein synthesis in rat liver^1–4. A high amino-acid concentration in the perfusion⁵ or incubation⁶ medium is needed to observe an effect of growth hormone in vitroon incorporation of precursors into protein and RNA of isolated hepatic tissue. We report here changes in the ability of cell-free systems to incorporate amino-acids into acid-insoluble material in vitrowhen they were prepared from slices incubated in various concentrations of amino-acids.     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Molecular Mechanisms of Actions of Interleukin-6 on the Brain,with Special Reference to Serotonin and the Hypothalamo-Pituitary-Adrenocortical Axis

Biological activities of the multifunctional cytokine, interleukin-6 (IL-6) include stimulation of B cell proliferation, immunoglobulin production, and initiation of the acute-phase response. IL-6 affects the CNS in that it activates the hypothalamo-pituitary-adrenocortical (HPA) axis and increases brain tryptophan and serotonin metabolism. IL-6 has been proposed as an important mediator of interaction between the neuroendocrine and immune systems. The peripheral and central effects of IL-6 are presumably mediated through its membrane receptor (IL-6R). IL-6, IL-6R and their respective mRNAs have been detected in several brain regions. Although the functions of cytokines overlap considerably, each displays its own characteristic properties. Expression of IL-6 in the brain has been observed in several CNS disorders, some of which have been associated with disorders of serotonin metabolism. It is proposed that interactions between IL-6 and brain serotonin is a complex process which involves corticotropin-releasing factor (CRF) and opioid peptides. It is likely that the molecular mechanisms underlying the actions of IL-6 on the HPA axis and its other brain functions involve the integrated effects of glutamate, Ca²⁺, 3,5-cyclic AMP, protein kinase C, and other metabolic pathways.     

<Emphasis Type="Italic">Withania coagulans</Emphasis> tryptophan decarboxylase gene cloning,heterologous expression,and catalytic characteristics of the recombinant enzyme

Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5′-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher K_m value for tryptophan than for pyridoxal 5′-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P_M, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P_M is produced at high pH with CO/O₂, no EPR-detectable radicals are formed. Conversion of the CO/O₂-generated P_M into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in P_M which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.     

Revised Amino-acid Sequences for Porcine and Human Adrenocorticotrophic Hormone

MEMBRANE protein of bovine rod outer segments has been studied by gel electrophoresis and amino-acid analysis. Membranes were purified in a sucrose density gradient¹ at an ionic strength below 0.001. The isolated material probably consisted of fragmented disk membranes¹. ‘Emulphogene’ solutions of rhodopsin were chromatographed on calcium phosphate²; the results for A₂₇₈: A₄₉₈ were 1.7–1.8, indicating good purity.     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.