Lyndon J. Rogers

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/μM gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of δ⇐inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects δ-aminolaevulinic acid synthesis rather than its subsequent metabolism.

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.

Purification and characterization of the vanadium bromoperoxidase from the macroalga Corallina officinalis

Abstract The red macroalga Corallina officinalis contains a bromoperoxidase whose activity is vanadium-dependent. This enzyme has been purified to homogeneity as judged from non-denaturing PAGE and SDS-PAGE. Sedimentation equilibrium studies gave a value for M r of 740 000, whereas a M r of 64 000 was obtained by SDS-PAGE. The native enzyme thus appears to be an oligomer, probably of 12 identical subunits. Kinetic studies for the bromination of monochlorodimedone (MCD) gave the following: K m Br− , 1.0 mM; K m H 2 O 2 , 60,μM and K m MCD , 21 μM. The bromoperoxidase showed remarkable thermostability, retaining appreciable activity at 60–70 ° and recovering the original activity on re-equilibration to 25°, even on repeated cycles of this temperature regime. The enzyme also showed tolerance to a range of organic solvents and chaotropic agents.

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 Å resolution: Description of the flavin mononucleotide binding site

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Photosynthesis and photosynthetic electron transport

The photosynthetic capacity of detached leaves of a non-yellowing mutant of Festuca pratensis Huds. declined during senescence at a similar rate to that in a normal cultivar. Respiratory oxygen uptake in the dark continued at similar rates in both genotypes during several days of senescence. In chloroplasts isolated from leaves at intervals after excision, the rate of photosystem I (PS I)-mediated methyl viologen reduction using reduced N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor also declined in both genotypes, possibly due to loss of integrity of the photosynthetic apparatus in the cytochrome f-plastocyanin region. There was a similar fall in PS II electron transport using water as electron donor and measured at the rate of reduction of 2,6-dichlorophenolindophenol. Partial restoration of this activity by the addition of diphenyl carbazide was evidence for lability of the oxygen-evolving complex during senescence. An accentuated difference between mutant and normal material in this case indicated that the mutant retains a greater number of functional PS II centres. Changes in the light-saturation characteristics of the two photosystems have been discussed in relation to the organization of the photosynthetic membranes during senescence.

R-phycoerythrin from the macroalgaCorallina officinalis (Rhodophyceae) and application of a derived phycofluor probe for detecting sugar-binding sites on cell membranes

R-Phycoerythrin (absorption spectrum 280; 495;sh 535, 564 nm with A564/A280 ratio of 5.3) was purified from the red macroalgaCorallina officinalis. The relative molecular mass determined from PAGE was 240 000. SDS-PAGE demonstrated two major subunits ofMr 20 000 and 21 000, respectively, and a minor subunit ofMr 30 000. A fucospyranosyl phenylisothiocyanate conjugate was prepared and this novel fluorescent affinity reagent used in conjunction with a flow cytometer to probe fucose-binding sites on blood mononuclear cells. By varying the sugar and using other phycobiliproteins the approach has the potential for simultaneously monitoring different sugar binding sites on subsets of cells within populations.

Metabolism of protein and chlorophyll in leaf tissue of Festuca pratensis during chloroplast assembly and senescence

Abstract Chlorophyll (chl) and protein were measured throughout greening and senescence of excised etiolated leaf tissue of Rossa, a normal cultivar of Festuca pratensis Huds., and of a mutant genotype, Bf993, which is unable to degrade pigments and associated thylakoid membrane proteins during foliar senescence. Bf993 and Rossa accumulated chl and proteins at the same rate during greening. On transferring leaf tissue to darkness after 48 hr illumination, chl continued to increase for a further 48 hr. Gabaculine, an inhibitor of chl synthesis, had only a small effect on the accumulation during darkness, suggesting that pre-existing porphyrin precursors, such as chlorophyllide, are utilized during this period. Prolonged incubation in darkness promoted senescence, during which chl was retained in the mutant as was the major chl-binding protein LHCP-2; cytochrome ƒ was also less labile in Bf993 than in Rossa. Two other thylakoid membrane proteins, protochlorophyllide oxidoreductase and the M r 33 x 10 3 protein of the oxygen-evolving complex of photosystem 2, proved equally labile in the mutant and wildtype during senescence. A common feature of the thylakoid membrane proteins exhibiting abnormal stability during senescence of mutant leaf tissue is that they incorporate prosthetic groups derived from tetrapyrroles. Since previous studies showed that the metabolic lesion in Bf993 seems to be sited in the pathway of chl catabolism immediately downstream of dephytylation, it is suggested that degradation of chl and other stabilizing tetrapyrroles, notably haem, provides the signal for breakdown of associated thylakoid apoproteins. The contrasting behaviour of Rossa and Bf993 tissue during greening and senescence supports the view that systems turning over pigment-proteolipid complexes during plastid assembly are not the same as those which degrade the complexes during senescence.

Endopeptidases during the development and senescence of Lolium temulentum leaves

The endopeptidase activity of Lolium temulentum leaf tissue was measured using azocasein as a substrate. The enzyme increased with leaf age, and also during senescence of excised leaf tissue. There were at least two distinct endopeptidase activities, characterized by different pH optima. The predominant form in leaves of intact plants was maximally active at pH 5. In detached leaves during the later stages of senescence this activity was replaced by an enzyme with an optimum at pH 8. An antibody raised against the non-glycosylated cysteine endopeptidase papain cross-reacted with polypeptides in protein preparations of L. temulentum leaf tissue. The correlation between enzyme activity and the pattern of immunoreactive polypeptides suggested that the polyclonal antibody was able to recognize the Lolium homologues of papain. The switch from pH 5 to pH 8 enzyme in detached leaves was associated with an evident decrease in the M, values of papain-like antigens detected on western immunoblots, from ca 60 000 to 30 000. It is possible that the alkaline activity is derived from the acid form, perhaps by limited autolysis in protein-depleted tissue at an advanced stage of senescence. On the other hand, the response of protease activation to treating leaf tissue with inhibitors of protein biosynthesis is more consistent with de novo appearance of a different form of the enzyme in late senescence.

A constitutive flavodoxin from a eukaryotic alga

Abstract We report the isolation and some properties of a flavodoxin from a eukaryotic organism, the naturally occurring red alga Chondrus crispus . Unlike the situation with most other organisms the flavodoxin, under normal growth conditions, is the predominantly formed low-potential electron carrier, an accompanying ferredoxin occurring in only very small amounts. The flavodoxin is of molecular weight 21000 and one mole of FMN is present per mole of protein. Reduction of the flavoprotein proceeds via a blue flavosemiquinone radical. The flavodoxin is active both in photosynthetic NADP reduction by broken chloroplasts, and in phosphoroclastic cleavage of pyruvate by cell-free extracts of Clostridium pasteurianum .

Cyanobacterial glutamate 1-semialdehyde aminotransferase: requirement for pyridoxamine phosphate

Glutamate 1-semialdehyde aminotransferase has been separated from metabolically related activities by gel filtration and affinity chromatography. The enzyme was inhibited by gabaculin, 4-amino 5-fluoropentanoic acid and pyridoxal 5-phosphate and stimulated by pyridoxamine 5-phosphate. The activity of enzyme recovered by elution after electrophoresis in non-denaturing polyacrylamide gels was wholly dependent on pyridoxamine 5-phosphate. A mechanism for the enzyme-catalysed reaction based on these observations is discussed.