Malnutrition, urocanic acid, and sun may interact to suppress immunity in sojourners to high altitude.

Irradiation of skin by ultraviolet radiation in mice and humans leads to a suppression of cell-mediated immunity. This process is initiated when one of the photoreceptors in skin, trans-urocanic acid, is photoisomerized to cis-urocanic acid, an immunomodulator. High levels of L-histidine, histamine, and trans-urocanic acid are found in humans and animals when they are protein malnourished. Mice fed on an elevated L-histidine diet have more trans-urocanic acid in the skin and are more susceptible to UV-induced immune suppression. Sojourners to high altitudes are malnourished, suffer protein catabolism, are exposed to sun, and often acquire infectious diseases. There is evidence that sunscreens may not adequately protect the immune system. Furthermore, UV intensity increases with altitude. We propose a testable hypothesis: UV radiation causes photoimmune suppression in sojourners to high altitude and this allows infectious diseases to develop. The mechanism we propose includes protein malnutrition, high levels of trans-urocanic acid, ultraviolet radiation, formation of cis-urocanic acid, immune suppression, and infection.

The degradation of L-histidine and trans- and cis-urocanic acid by bacteria from skin and the role of bacterial cis-urocanic acid isomerase.

UV-B radiation suppresses cell-mediated immunity. Histidine forms trans-urocanic acid (trans-UCA) enzymatically in the stratum corneum. Photoisomerization of trans-UCA to cis-urocanic acid (cis-UCA) has been proposed for the initiation of an immunosuppressive process. Many microorganisms described in the literature metabolize histidine and/or trans-UCA. Our enrichment cultures of soil and sewage contain organisms that can degrade cis-UCA. We have tested microorganisms for degradation of cis-UCA, trans-UCA, or L-histidine when they are incorporated at 0.2% in nutrient broth. Six out of 10 selected genera isolated by our clinical microbiology laboratory degrade one or more of the imidazole substrates. We have cultured over 60 aerobic isolates from human skin. Of these, 33 degrade one or more of the three imidazole substrates and 12 degrade cis-UCA. Isolates from BALB/c mice are also active on cis-UCA. We have identified a cis-UCA-degrading bacterium as Micrococcus luteus. Four ATCC strains of M. luteus have been tested and three are active on histidine or trans-UCA; two are active on cis-UCA. Micrococci that degrade cis-UCA contain a new enzyme, cis-UCA isomerase, which converts the substrate to the trans-isomer. This enzyme provides access to the classical L-histidine degradation pathway. We hypothesize that an epidermal microflora that degrades L-histidine, trans-UCA, or cis-UCA influences the concentration of urocanic acids on the skin and, thus, affects immune suppression.

The potential role for urocanic acid and sunlight in the immune suppression associated with protein malnutrition.

Irradiation of skin by sunlight or ultraviolet B (UVB, 290-320 nm) brings about a downregulation of cell-mediated immunity. An action spectrum for photoimmune suppression in mice indicates that trans-urocanic acid absorbs UV photons and is isomerized to the cis-isomer in the stratum corneum. Cis-urocanic acid is subsequently shown to suppress cellular immunity in mice. When histidine is elevated in a mouse diet, a higher level of urocanic acid is detected in mouse skin. These mice are more susceptible to photoimmune suppression. There is evidence that humans and animals experiencing protein malnutrition have very high levels of urocanic acid and/or histidine. Urocanic acid is formed by deamination of histidine in one enzymatic step. We discuss the protein malnutrition of kwashiorkor patients. They experience suppressed immunity and disturbed histidine metabolism. Here, we present a testable hypothesis: one cause of the immune deficiency observed in humans with protein malnutrition is the photoconversion by UVB of increased levels of trans-urocanic acid in skin to cis-urocanic acid, which suppresses the cellular immune system.

Adventitious interconversion of cis- and trans-urocanic acid by laboratory light.

Urocanic acid (UCA) is a chromophore in the stratum corneum. Ultraviolet radiation (ultraviolet B) has been shown to suppress mammalian cell-mediated immunity. The photoisomerization of trans-UCA to cis-UCA was proposed as the initiator of the suppression process. Cis-urocanic acid has been demonstrated to suppress immunity by a variety of experiments. Investigators should be aware that laboratory illumination may be capable of interconverting trans-UCA and cis-UCA during experimental manipulations. This possible inadvertent contamination of one isomer by the other may influence results. We demonstrated that fluorescent lamps, daylight, sunlight and incandescent lamps were able to bring about isomerization. Window glass and container materials of plastic and clear glass did not filter out effective wavelengths, but three commercial plastic diffusers on fluorescent fixtures prevented the isomerization. Because the molar extinction coefficient (epsilon) for cis-UCA is less than that of trans-UCA, we have exposed 0.1 mM trans-UCA to ambient light and monitored the change in absorbance. A method is given to calculate the percentage of trans and cis isomers from the absorbance at 277 nm when the initial purity and absorbance are known. Using this procedure, we validated the molar extinction coefficient of cis-UCA.

Photomodulation of enzymes.

The photomodulation of enzymes involves the activation and inactivation of enzyme reactions by UV and visible light. Enzymes or their reactions may be affected directly or indirectly. Direct effects involve photoproduction of a substrate, photodissociation of an inhibitor, photochemistry of protein amino acids, irradiation of a chromophore and irradiation of an enzyme substrate. Indirect effects involve gene expression, phytochrome and other photoreceptors which are not part of the enzyme, protein synthesis, membranes and photosynthesis. Photoactivation of enzymes is related to photocarcinogenesis, photomorphogenesis of plants, primary effects or side effects of phototherapy, deoxyribose nucleic acid (DNA) repair and many other aspects of biology and medicine. Model systems may contribute to the knowledge of protein chemistry and medicinal chemistry.

Thermal activation of photoactivatable urocanase from Pseudomonas putida.

The dark inactivation of urocanase from Pseudomonas putida is caused by the formation of a sulfite adduct of the tightly bound coenzyme, nicotinamide adenine dinucleotide. Photodissociation of this adduct by UV radiation restores the enzyme activity. Based on cold exhaustive dialysis the modification reaction appeared to be irreversible. However, we now report that sulfite modification of urocanase is reversible at higher temperatures. An Arrhenius plot of the thermal activation is linear (20-38 degrees C). The activation energy for the enzyme activation is 114 kJ mol-1. The substance that is photodissociated from inactive urocanase reacts with urocanase to reform the modified enzyme indicating that sulfite is not oxidized, or otherwise changed through these processes. Nucleophiles (sulfite, hydroxylamine, hydride, cyanide) are known to inhibit urocanase by forming adducts with nicotinamide adenine dinucleotide. Urocanase inactivated by hydride or cyanide is not reactivated thermally or photochemically. Urocanase inactivated by hydroxylamine and by glycylglycine can be reactivated by a thermal reaction. In conclusion, sulfite-modified urocanase, which is formed in cells, can be reactivated not only by sunlight but also at physiological temperatures.

Specific inhibition of bacterial and bovine urocanases by glycylglycine.

Urocanase (EC 4.2.1.49) purified from Pseudomonas putida was unexpectedly inhibited by the dipeptide glycylglycine. Using a spectrophotometric assay for urocanase activity, we characterized the inhibition. The inhibition was temperature-, concentration-, and time-dependent; 0.1, 0.5 and 1.0 mM glycylglycine inhibited the enzyme by 20%, 50% and 78%, respectively, in 60 min at 30 degrees C. Dithiothreitol and reduced glutathione did not prevent the process. The inhibition was a pseudo first-order reaction. Three ligands that bind to the active site, urocanate, imidazole-propionate (a competitive inhibitor) and sulfite, protected the enzyme from glycylglycine inhibition. The inhibition was very specific for glycylglycine, because fifteen related biochemicals, including glycine, triglycine, and tetraglycine, were not effective. Ethylenediaminetetraacetic acid and other chelators did not inhibit urocanase. Bovine liver urocanase was also inhibited by this peptide. The characteristics of this inhibition suggest that glycylglycine acts at the active site, does not function by metal binding and that minor alterations in the glycylglycine molecule preclude the inhibition. A specific inhibition of urocanases by glycylglycine has been observed.

Activation of urocanase from Pseudomonas putida by electronically excited triplet species.

Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and, as shown previously, is activated by the direct absorption of ultraviolet light. In this study, we describe the activation of urocanase by energy transfer from triplet indole-3-aldehyde, generated in the peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. The activation was time-, temperature-, and pH-dependent. The involvement of reactive oxygen intermediates was excluded by the lack of effect of appropriate quenchers and traps. Triplet quenchers, in contrast, reduced the level of activation. Photoexcited rose bengal, a triplet species of a different nature and origin, was also effective in promoting activation. These results demonstrate a potential mechanism of urocanase regulation not dependent on an environmental source of light, but rather brought about by an enzymically generated excited species.

Evidence against a temperature-dependent conformational change in urocanase from Pseudomonas putida.

Enzymatic activity of urocanase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) has an unusual resistance to temperature changes, and a temperature-dependent conformational change has been suggested (Hug, D.H. and Hunter, J.K. (1974) Biochemistry 13, 1427-1431). A conformational change or dissociation has been proposed in the range of 29-31 degrees C (Cohn, M.S., Lynch, M.C. and Phillips, A.T. (1975) Biochim. Biophys. Acta 377, 444-453). In this work, no evidence was found for a temperature-dependent conformational change or dissociation. Arrhenius plots of Km and Vmax were linear; the sedimentation coefficient was independent of temperature; tryptophanyl fluorescence was a linear function of temperature; and heat capacity calorimetry showed no transitions below 60 degrees C.

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.