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Biomedical subjects

P A Castelfranco

Publications and source records attributed to P A Castelfranco.

At least 37 records · Page 2Linked to original sources
PubMed1984

Localization of Mg-Chelatase and Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Activities within Isolated, Developing Cucumber Chloroplasts.

Magnesium chelatase and magnesium protoporphyrin IX monomethyl ester (oxidative) cyclase activities were both sensitive to inhibition by p-chloromercuribenzoate in intact, developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts. Magnesium chelatase was also sensitive to the membrane-impermeable mercurial p-chloromercuribenzene sulfonate (PCMBS), while cyclase activity was only slightly sensitive. When the plastids were pretreated with PCMBS, triosephosphate dehydrogenase activity was inhibited very slightly, indicating that PCMBS does not readily penetrate through the chloroplast envelope. These results suggest that magnesium chelatase is located in the chloroplast envelope, while the cyclase is located deeper within the chloroplast.

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PubMed1983

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts.

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.

Aerobiosis↗
PubMed1983

Separation of monovinyl and divinyl protochlorophyllides and chlorophyllides from etiolated and phototransformed cucumber cotyledons.

A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0 degrees C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark.

Journal Article↗
PubMed1982

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS.

Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with delta-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.These results are consistent with the presence of an O(2), Fe-requiring step between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide.

Journal Article↗
PubMed1982

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. OBSERVATIONS ON THE BIOSYNTHETIC PATHWAY IN ISOLATED ETIOCHLOROPLASTS.

The conversion of l-glutamate to delta-aminolevulinate, in preparations of cucumber etiochloroplasts incubated in vitro, was inhibited by protoheme IX and Mg-protoporphyrin IX. Mg-protoporphyrin IX was destroyed in the presence of air and plastids; this breakdown was accelerated by S-adenosyl methionine. Mg-protoporphyrin IX was also converted to protochlorophyllide in vitro. This conversion exhibited an absolute requirement for atmospheric oxygen and was strongly stimulated by S-adenosyl methionine and by darkness.Based on these results, and on the results of the preceding paper (Spiller, Castelfranco, Castelfranco 1981 Plant Physiol 68: 107-111), a comprehensive hypothesis for the role of O(2) and Fe in chlorophyll biosynthesis is formulated.

Journal Article↗
PubMed1982

Separation of Mg-Protoporphyrin IX and Mg-Protoporphyrin IX Monomethyl Ester Synthesized de novo by Developing Cucumber Etioplasts.

High pressure liquid chromatography was used to demonstrate that chelation of Mg(2+) into protoporphyrin IX precedes methylation in isolated greening etioplasts from cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. Mg-protoporphyrin IX synthesized in vitro from protoporphyrin IX, Mg(2+), and ATP or exogenous Mg-protoporphyrin IX could serve as substrates for the methylation step. In either case, S-adenosylmethionine was the methyl donor and could not be replaced by ATP plus methionine.

Journal Article↗
PubMed1982

In Vitro Synthesis of the Chlorophyll Isocyclic Ring : Transformation of Magnesium-Protoporphyrin IX and Magnesium-Protoporphyrin IX Monomethyl Ester into Magnesium-2,4-Divinyl Pheoporphyrin A(5).

Developing chloroplasts of Cucumis sativus L., cv Beit Alpha which were incubated with either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester in darkness produced a partially phototransformable protochlorophyllide species that was tentatively identified as Mg-2,4-divinyl pheoporphyrin a(5). S-Adenosylmethionine stimulated Mg-2,4-divinyl pheoporphyrin a(5) formation irrespective of the starting material used. In the case of Mg-protoporphyrin IX monomethyl ester, this stimulation was attributed to the need to remethylate substrate that had been hydrolyzed by an endogenous methylesterase which converts part of the added Mg-protoporphyrin IX monomethyl ester to Mg-protoporphyrin IX.NADP and NADPH stimulated the conversion of Mg-protoporphyrin IX to Mg-2,4-divinyl pheoporphyrin a(5). The conversion required oxygen and was half saturated at 50 micromolar dissolved O(2). The conversion was insensitive to inhibitors of iron-sulfur and heme-containing proteins, to Cu chelators, H(2)O(2), and peroxide scavengers. However, the conversion was extremely sensitive to phenazine methosulfate, methylene blue, and methyl viologen.A decrease of the plastids' ability to convert Mg-protoporphyrin IX to Mg-2,4-divinyl pheoporphyrin a(5) after lysis in 0.1 molar NaCl suggested a requirement for plastid integrity.

Journal Article↗
PubMed1981

Properties of Magnesium Chelatase in Greening Etioplasts: METAL ION SPECIFICITY AND EFFECT OF SUBSTRATE CONCENTRATIONS.

Evidence was obtained by means of low temperature spectrofluorimetry and high pressure liquid chromatography for the exclusive chelation of Mg(2+) into protoporphyrin IX by a plastid preparation extracted from greening cucumber cotyledons. Under the experimental conditions which were optimized for Mg(2+) chelation, Zn(2+) chelation was not detected. However, Zn(2+) chelation was observed when ATP was omitted and ZnCl(2) was added to the incubation mixture. The observed Zn(2+) chelation was partially nonenzymic. The requirements for the enzyme, Mg chelatase, are discussed in terms of the regulatory function of this enzyme.

Journal Article↗
PubMed1980

ATP requirement for mg chelatase in developing chloroplasts.

The synthesis of Mg-protoporphyrin-IX from exogenous protoporphyrin-IX, in a crude plastid pellet extracted from greening cucumber cotyledons was found to require l-glutamate as a cofactor. It has now been shown that glutamate acts in the presence of contaminating mitochondria to provide an ATP regenerating system. With purified plastids, Mg chelatase is not stimulated by glutamate; instead, it requires a high concentration of ATP and is greatly stimulated by added phosphoenolpyruvate and pyruvate kinase. GTP, UTP, CTP, and ITP will not substitute for ATP. ADP in the absence of an ATP generating system is completely ineffective, whereas it is slightly inhibitory in the presence of 10 mm ATP. AMP is strongly inhibitory in the reaction; 50% inhibition is obtained at approximately 3.5 mm AMP in the presence of 10 mm ATP.

Journal Article↗
PubMed1975

Protoheme turnover and chlorophyll synthesis in greening barley tissue.

Studies in which (14)C-labeled precursors were fed to etiolated barley leaves (Hordeum vulgare L. var. Proctor) yielded chlorophyll and protoheme having similar specific radioactivities. These findings indicate: (a) there appears to be a rapid turnover of protoheme in the absence of net synthesis; (b) both pigments probably originate from a single 5-aminolevulinic acid pool; (c) the efficient utilization of glutamate-1-(14)C and the relatively poor utilization of glycine-2-(14)C suggest that 5-aminolevulinic acid is probably synthesized by a pathway other than 5-aminolevulinic acid synthetase (succinyl CoA-glycine succinyltransferase) in agreement with previously published work; (d) protoheme turnover appears to be faster under conditions which allow for rapid chlorophyll accumulation; (e) difference spectra indicate that mitochondrial cytochromes make a relatively minor contribution to the total heme in barley leaves. These findings are discussed in the light of current knowledge about tetrapyrrole regulation in photosynthetic organisms.

Journal Article↗
PubMed1974

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues.

delta-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. delta-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of delta-aminolevulinic acid dehydrase. The rate of delta-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of delta-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, delta-aminolevulinic acid accumulation ceased.delta-Aminolevulinic acid accumulation showed an absolute requirement for oxygen and was inhibited drastically by cyanide and azide, and to a lesser extent by arsenite and malonate. 2,4-Dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, sodium fluoroacetate, and hydroxylamine hydrochloride showed no effect under the conditions tested. Freezing and thawing of the tissue completely prevented the accumulation of delta-aminolevulinic acid.The findings of this investigation are consistent with the hypothesis that delta-aminolevulinic acid is a chlorophyll precursor in higher plants, and that chlorophyll biosynthesis is regulated at the level of the formation of delta-aminolevulinic acid.

Journal Article↗
PubMed1974

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues.

delta-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various (14)C-labeled precursors and levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydrase. The radioactivity in the accumulated delta-aminolevulinic acid was measured.The most effective labeled precursors were the 5 carbon dicarboxylic compounds glutamate, glutamine, and alpha-ketoglutarate. (14)C-Labeled glycine and succinate were relatively poor. The carboxyl and the methylene carbons of glycine were incorporated into delta-aminolevulinic acid to about equal extent. The carboxyl carbon of glutamate was incorporated almost as well as the internal carbons of the same compound. These results are inconsistent with the succinyl CoA-glycine succinyl transferase (delta-aminolevulinic acid synthetase) mode of delta-aminolevulinic acid production.When the same experiments were performed on turkey blood (which, as avian blood in general, possesses delta-aminolevulinic acid synthetase), delta-aminolevulinic acid was labeled most effectively from glycine-2-(14)C, moderately well from glycine-1-(14)C and glutamate-3,4-(14)C and not at all from glutamate-1-(14)C.It appears probable that greening higher plant tissues possess an alternate route to delta-aminolevulinic acid in which the carbon skeleton of glutamate (and alpha-ketoglutarate) is incorporated intact into the first committed metabolite of the chlorophyll pathway.

Journal Article↗
PubMed1974

The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta-Aminolevulinic Acid.

Etiolated cucumber cotyledons treated with delta-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the delta-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.The treatment which effects lag phase removal in control tissue did not affect the course of chlorophyll (ide) accumulation in delta-aminolevulinic acid-treated tissue under either continuous or intermittent illumination. It was concluded that the lag phase in etiolated tissues must reflect the gradual development of the ability to synthesize delta-aminolevulinic acid at an appreciable rate.

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