Albert Kahn

tRNAGlu as a cofactor in δ-aminolevulinate biosynthesis: steps that regulate chlorophyll synthesis

Abstract In plants δ-aminolevulinate is formed from the intact carbon skeleton of glutamate catalysed by the action of three enzymes. The first step of the pathway is activation of glutamate by ligation to δ-ALA-RNA, a reaction identical to that in protein synthesis. Intriguingly, this RNA has been identified as the chloroplast tRNA Glu .

Biosynthesis of Δ-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase

Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

Genetic regulation of chlorophyll synthesis analyzed with mutants in barley.

Barley seedlings homozygous both for the xantha-135 and tigrina-d12 mutation accumulate magnesium protopophyrins and other precursors of chlorophyllide constitutively in darkness. The homozygous double mutant xantha-f10, tigrina-O34 produces protoporphyrin constitutively. These results provide evidence for the control of chlorophyllide synthesis in higher plants through the products of regulatory genes in the nucleus.

Photoconvertible protochlorophyll(ide)635650 in vivo: A single species or two species in dynamic equilibrium?

Abstract The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.

Kinetics and quantum yield of photoconversion of protochlorophyll(ide) to chlorophyll(ide) a

Abstract The kinetics of photoconversion of protochlorophyll(ide) to chlorophyll(ide) a were investigated in dark-grown barley leaves and in a preparation of protochlorophyll holochrome subunits. In the subunits the conversion obeyed first-order kinetics. This indicates that the excitation of protochlorophyll(ide), energy loss through deexcitation, and the reduction of excited protochlorophyll(ide) are all reactions that follow first-order kinetics with respect to protochlorophyll(ide) in protochlorophyll holochrome subunits. In contrast, photoconversion in leaves obeyed neither first- nor second-order kinetics. This prompted the postulation of an additional route within macromolecular units of protochlorophyll holochrome, whereby energy is lost from excited protochlorophyll(ide) by a reaction that is not first order. Such a process might be energy transfer from excited protochlorophyll(ide) to newly-formed chlorophyll(ide) a. A dynamic model describing photoconversion in macromolecular units was derived. The model is consistent with the observed progress of photoconversion in barley leaves and in protochlorophyll holochrome subunits from barley. Determinations of the quantum yield of photoconversion in protochlorophyll holochrome subunits gave values of 0.4–0.5 molecules · quantum−1. Estimates of the initial quantum yield of the photoconversion process in leaves fell into the same range. The dynamic model allows predictions on the progressively decreasing quantum yield as the photoconversion proceeds in macromolecular units.

Gabaculine-resistant mutants of Chlamydomonas reinhardtii with elevated glutamate 1-semialdehyde aminotransferase activity

Gabaculine-resistant mutants were obtained from UV-irradiated Chlamydomonas reinhardtii by selection on agar plates containing 2 or 4mm-gabaculine. Genetic analysis of two of the mutants showed that the mutations conferring gabaculine resistance lie in non-allelic nuclear genes. These genes,gab 1 andgab 2 are linked to their respective centromeres and not to each other. Glutamate 1-semialdehyde aminotransferase activity in the soluble protein fraction of these mutants and a third one was 1.5 to 4 fold higher than in the wild type. Gabaculine inhibited the aminotransferase activity in the extracts from wild type and all three mutant strains of algae.

Polypeptide composition of internal membranes from barley etioplasts

The polypeptides of isolated etioplast membranes and chloroplast membranes were solubilized in sodium dodecyl sulfate. Protein band patterns were obtained by electrophoresis on polyacrylamide slab gels with separating gels containing either a uniform polyacrylamide concentration or a concentration gradient. Several characteristic chloroplast bands were absent or reduced in intensity in patterns of etioplast membranes. Conversely, certain bands found in etioplast membrane patterns were reduced or absent in patterns of chloroplast membranes. Among these was a distinct doublet detected only in patterns of etioplast membrane polypeptides obtained with gels lacking urea. Some bands, for example those tentatively identified as subunits of coupling factor were found in patterns of etioplast as well as chloroplast membranes.

Circular dichroism of protochlorophyllide and chlorophyllide holochrome subunits

Dark-grown seedlings of angiosperms accumulate protochlorophyll(ide) (Pchl), which upon illumination, is reduced to chlorophyll(ide) a (Chl). A photoactive pigment-protein complex, protochlorophyll holochrome (P&l-H) [ 1,2] with macromolecules of mol. wt. about 600 000 [3] containingmultiplePchl molecules per particle [4, 51 can be extracted from etiolated bean (Phase&s vulguris L.) leaves, with the aid of a detergent mixture of saponins, P&l-H subunits with an apparent mol wt of 63 000 and giving no indication of more than one Pchl molecule per unit have been obtained from dark-grown barley (Hordeum vulgure L.) leaves [6,7]. Exciton interaction among closely associated pigment molecules in Pchl-H from bean leaves and its photoproduct, chlorophyll holochrome (Chl-H) has been inferred, from circular dichroism (CD) spectra in the red region [8,9]. Previous CD work in the region from 540 to 615 nm on saponin-containing Pchl-H from barley [lo] does not allow comparison with the CD results on bean. This paper gives the results of a comparative study of CD spectra in the red region gained from a number of saponin-containing preparations of Pchl-H and Chl-H from dark-grown barley and bean leaves. In general, we did not detect splitting of Pchl or Chl