Formation of a photoreversible phycocyanobilin-apophytochrome adduct in vitro.

Avena seedlings grown in the presence of the plant tetrapyrrole synthesis inhibitor 4-amino-5-hexynoic acid contain less than 10% of the spectrally detectable phytochrome levels found in untreated seedlings, but continue to accumulate phytochrome apoprotein (Elich, T. D., and Lagarias, J. C. (1988) Plant Physiol. 88, 747-751). Using such tetrapyrrole-deficient seedlings, we have previously reported that phycocyanobilin, the cleaved prosthetic group of C-phycocyanin, can be incorporated into phytochrome in vivo to yield spectrally active holoprotein (Elich, T. D., McDonagh, A. F., Palma, L. A., and Lagarias, J. C. (1988) J. Biol. Chem. 264, 183-189). Here we show that addition of phycocyanobilin to soluble extracts of inhibitor-treated seedlings results in a rapid increase in spectrally active phytochrome holoprotein. The newly formed photoactive species displays a blue-shifted absorbance difference spectrum similar to that observed in the previous in vivo studies. The increase in spectral activity is consistent with conversion of all of the preexisting phytochrome apoprotein to functionally active holoprotein. The formation of a covalent phycocyanobilin-apophytochrome adduct is shown by an increase in Zn2+-dependent bilin fluorescence of the phytochrome polypeptide. A photoreversible, covalent adduct with a similar optical spectrum also forms when immunopurified apophytochrome is incubated with phycocyanobilin. ATP, reduced pyridine nucleotides, or other cofactors are not required for adduct formation. When biliverdin IX alpha is substituted for phycocyanobilin, no spectrally active covalent adduct is produced. These results indicate that an A-ring ethylidene-containing bilatriene is required for post-translational covalent attachment of bilin to apophytochrome and that apophytochrome may be the bilin C-S lyase which catalyzes bilin attachment.

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.

Comparative neurotoxicity and pyrrole-forming potential of 2,5-hexanedione and perdeuterio-2,5-hexanedione in the rat.

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, reacts with protein amines to form alkylpyrrole adducts. Pyrrolylation of neurofilament protein may be the initiating molecular event in 2,5-HD neuropathy. The present study compares the neurotoxic and pyrrole-forming potentials of 2,5-HD with those of perdeuterio-2,5-HD ([D10]-2,5-HD) in the rat. Due to a requirement for C-H bond breaking in the reaction mechanism, the latter derivative was expected to exhibit a primary isotope effect, thus forming the pyrrole at a slower rate. In vitro studies confirmed that [D10]-2,5-HD pyrrolylated protein at only one-third of the initial rate seen with native 2,5-HD. Prolonged incubation resulted in similar pyrrole concentrations with both derivatives. Adult, male Wistar rats were administered daily (5 days/week) ip doses of either 3.5 mmol 2,5-HD or [D10]-2,5-HD/kg/day for 17 days or 2.5 mmol/kg/day for 38 days. At termination, animals administered 2,5-HD and [D10]-2,5-HD exhibited 27 and 8% body weight loss, respectively. Moderate to severe hindlimb paralysis was present in the 2,5-HD groups while only mild effects were seen in [D10]-2,5-HD-dosed rats. Neuropathological changes were prominent in spinal cord sections from 2,5-HD-treated animals, while no effects were present in rats given the deuterated derivative. Pyrrole adduct concentrations in serum and axonal cytoskeletal proteins from 2,5-HD-treated animals were two- to threefold higher than in rats given equimolar doses of [D10]-2,5-HD. Levels of covalent crosslinking of axonal cytoskeletal proteins (assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) appeared to correlate with pyrrole concentrations. Tissue concentrations of each diketone isomer were not significantly different, indicating similar uptake of native and deuterated 2,5-HD. Mass spectrometry revealed rapid back exchange of the terminal (methyl) but not of the internal (methylene) deuteriums of [D10]-2,5-HD in vivo. These findings support an absolute requirement for pyrrole formation in gamma-diketone neurotoxicity.

An efficient and prolonged in vitro translational system from isolated cucumber etioplasts.

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursors protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immunoprecipitation with anti-spinach RuBisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.

Regional distribution in rat brain of 1-pyrroline-5-carboxylate dehydrogenase and its localization to specific glial cells.

A possible alternative route for production of a small glutamate pool in brain is from proline or ornithine to 1-pyrroline-5-carboxylate (P5C) and thence to glutamate. The conversion from ornithine to P5C is catalyzed by ornithine delta-aminotransferase (OrnT) whereas that from proline is catalyzed by proline oxidase (PrO). The conversion of P5C to glutamate is catalyzed by 1-pyrroline-5-carboxylate dehydrogenase (PDH). Biochemical assays of PDH and PrO in various rat brain regions indicate no positive correlation between the two enzymes nor between either activity and high-affinity glutamate uptake or the regional distribution of OrnT. We have localized PDH and PrO histochemically by modifications of the Van Gelder [J. Neurochem. 12, 231-237, (1965)] method for gamma-aminobutyric acid (GABA) transaminase. The enzymes were found only in certain types of glial cells; the best stained were the Bergmann glial cells of the cerebellum but, for PDH, there was also good staining of astrocytes in the dentate area of the hippocampus. Since both these areas are believed to have heavy glutamate innervation and numerous GABA interneurons, these findings may reflect an alternative route of glutamate production in glial cells near some glutamate and/or GABA tracts but they do not support this as a possible route for glutamate formation in most brain regions. The findings do, however, provide further evidence for chemical specialization of glial cells.

[Biosynthesis of corrinoids and other tetrapyrrole compounds by an acetogenic Clostridium]. / Biosintez korrinoidov i drugikh tetrapirrol'nykh soedinenii atsetogennym klostridiem.

Biosynthesis of corrinoids and other tetrapyrrole pigments by the pure culture of the acetogenic Clostridium 99 was studied. When growing on media containing glucose or methanol, the physiological and biochemical characteristics of Clostridium 99 are very close to those of C. thermoautotrophicum. Methanol was shown to stimulate the corrinoid accumulation with the yield increasing from 154 micrograms/g dry biomass (glucose medium) up to 2250 micrograms/g dry biomass (methanol medium). According to the paper chromatography the corrinoid accumulated in Clostridium 99 cells differed both from vitamin B12 and Factor III. A study on the composition of extracellular tetrapyrroles, accumulated when the culture grows on the medium containing glucose and delta-aminolevulinic acid, revealed that they are represented both by uroporphyrin III and sirohydrochlorine-like pigments. The latters differ by a number of properties from sirohydrochlorine (corrifirine-2) of propione acidic bacteria. These pigments appear to be involved as intermediants in biosynthesis of corrinoids and other tetrapyrroles.

Biosynthesis of the chromophore of phycobiliproteins. A study of mesohaem and mesobiliverdin as possible intermediates and further evidence for an algal haem oxygenase.

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium.

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase high-pressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.

Pyrrolomycins F1, F2a, F2b and F3, new metabolites produced by the addition of bromide to the fermentation.

New antibiotics, pyrrolomycins F1, F2a, F2b and F3 were produced by Actinosporangium vitaminophilum sp. nov. when bromide ion was added to the fermentation medium. Addition of other halide ions such as chloride, iodide and fluoride ion showed no effect on pyrrolomycin production, affording polychlorinated pyrrolomycins A, B, C, D and E, but no F components. All four new antibiotics contain 2 approximately 4 mol of bromine, and their substitutional position was determined by X-ray analysis and synthesis, supported by spectroscopic analysis. They are strongly active against Gram-positive bacteria and fungi.

Factors influencing pyrroline 5-carboxylate synthesis from glutamate by rat intestinal mucosa mitochondria.

Factors influencing pyrroline 5-carboxylate (P5C) synthesis from glutamate by a subcellular fraction enriched in mitochondria of rat small intestinal mucosa have been studied. P5C synthesis decreased rapidly if this subcellular fraction was preincubated at 20 degrees C in the absence of substrates; this effect suggests that the enzyme(s) catalyzing P5C synthesis from glutamate (P5C synthase) is unstable in the absence of substrates. In the presence of substrates P5C synthesis increased linearly for the first 30 min of incubation, suggesting that the substrates promote enzyme stability. Pyridoxal 5'-phosphate is an effective inhibitor of P5C synthase whereas pyridoxamine 5'-phosphate and pyridoxal are not inhibitory. Potassium phosphate, KCl, and KBr each inhibited P5C synthase but potassium-Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) did not. Potassium phosphate was the most potent inhibitor followed by KBr, and then KCl. These results suggest P5C synthase is sensitive to anion inhibition. Both L-ornithine and D-ornithine inhibited P5C synthase; L-proline did not inhibit. Several analogs of ornithine and proline were also tested and none was found to inhibit P5C synthase; the inhibition by ornithine is, therefore, rather specific and it may prove to contribute to the regulation of metabolism of these amino acids.

[Microbiological synthesis of tetrapyrrole compounds]. / Mikrobiologicheskii sintez tetrapirrol'nykh soedinenii.

This paper reviews publications on the biosynthesis of functional tetrapyrroles by microorganisms. Emphasis is given to the structure of uroporphyrin III methylated derivatives termed corriphyrins and their involvement in the formation of two groups of tetrapyrrole pigments--corrinoids and siroheme. Current concepts concerning the final stages of the formation of the corrine ring and potential cobalt-free precursors of vitamin B12 are discussed. It is indicated that the data available may help elucidate evolutionary and biogenetic patterns in the emergence and interaction of tetrapyrrole compounds and formulate problems of practical importance.

The use of N-methylprotoporphyrin dimethyl ester to inhibit ferrochelatase in Rhodopseudomonas sphaeroides and its effect in promoting biosynthesis of magnesium tetrapyrroles.

N-Methylprotoporphyrin dimethyl ester inhibits ferrochelatase in isolated membranes of Rhodopseudomonas sphaeroides at low concentrations (around 10 nm). Full inhibition developed after a short lag phase. The inhibition was non-competitive with porphyrin substrate. Addition of inhibitor to growing cultures of Rps. sphaeroides caused a decrease (near 40%) in cytochrome content and a severe inhibition of ferrochelatase; the excretion of haem into the medium by cell suspensions was also severely inhibited. The addition of N-methylprotoporphyrin dimethyl ester to suspensions of photosynthetically competent Rps. sphaeroides Ga caused excretion of Mg-protoporphyrin monomethyl ester. When added to mutants V3 and O1, magnesium divinylphaeoporphyrin a5 monomethyl ester and 2-devinyl-2-hydroxyethylphaeophorbide a were excreted, with maximum effect at around 3 microM-inhibitor in the medium. The results are interpreted to suggest that the inhibitor decreases concentration of intracellular haem, which normally controls the activity of 5-aminolaevulinate synthetase. Unregulated activity of this enzyme leads to overproduction of protoporphyrin, which is diverted to the bacteriochlorophyll pathway. Further control operates at magnesium protoporphyrin ester conversion in normal cells.

The effect of N-methylprotoporphyrin IX on the synthesis of photosynthetic pigments in Cyanidium caldarium. Further evidence for the role of haem in the biosynthesis of plant billins.

N-Methylprotoporphyrin IX strongly inhibits synthesis of phycocyanobilin, but not chlorophyll a, in the dark. In the light, both phycocyanin and chlorophyll a synthesis are inhibited in parallel. These results are consistent with the intermediacy of haem in algal bilin synthesis and suggest a control mechanism for chlorophyll a synthesis, previously unknown.

Function of plasmids in the production of aureothricin. I. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis.

The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin. An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a. Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. From these results, the following conclusions were obtained. First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration. Second, the structural genes for biosynthesis of AT probably exist on chromosome. Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.

Mechanism of bile-pigment synthesis in algae. 18O incorporation into phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium.

The origin of the lactam oxygen atoms of phycocyanobilin from Cyanidium caldarium was studied using 18O labelling. By inhibiting photosynthesis, a high 18O enrichment was maintained in the gas phase and the resulting incorporation of label showed that the lactam oxygen atoms were derived from two oxygen molecules. Slow exchange of these oxygen atoms with water was demonstrated directly by using H218O.