Harmonic generation by yeast cells in response to low-frequency electric fields.

We report on harmonic generation by budding yeast cells (Saccharomyces cerevisiae, 10(8) cells/ml) in response to sinusoidal electric fields with amplitudes ranging from zero to 5 V/cm in the frequency range 10-300 Hz. The cell-generated harmonics are found to exhibit strong amplitude and frequency dependence. Sodium metavanadate, an inhibitor of the proton pump known as H+-ATPase, and glucose, a substrate of H+-ATPase, are found to increase harmonic production at low amplitudes while reducing it at large amplitudes. This P-type proton pump can be driven by an oscillatory transmembrane potential, and its nonlinear response is believed to be largely responsible for harmonic production at low frequencies in yeast cells. We find that the observed harmonics show dramatic changes with time and in their field and frequency dependence after perturbing the system by adding an inhibitor, substrate, or membrane depolarizer to the cell suspension.

Oxidatively damaged proteins of heart mitochondrial electron transport complexes.

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.

C(5)-C(5a)-modified bicyclomycins: synthesis, structure, and biochemical and biological properties.

Bicyclomycin (1) is a novel antibiotic that targets rho transcription termination factor in Escherichia coli. We have demonstrated that retention of the C(5)-C(5a) exomethylene unit in 1 is not essential for inhibition. In a recent paper we proposed a working model for 1 and rho function and suggested that 1 binds in a cleft with the C(5)-C(5a) exomethylene unit directed toward the dimeric interface of two rho monomers. This report examines the bicyclomycin C(5)-C(5a) structural constraints necessary for retention of rho inhibitory activity. Three classes of C(5)-C(5a)-modified bicyclomycins have been prepared and their inhibitory activities evaluated in the poly C-dependent ATPase and filter disk antimicrobial assays. The first series consisted of 12 analogues (8-19) that contained a C(5a)-unsaturated substituent and possessed C(5E)-geometry. The second set were a pair of C(5a)-substituted C(5E)- and C(5Z)-geometrical isomers (21 and 23). The final group of compounds consisted of six C(5)-C(5a)-dihydrobicyclomycins (24-28, 34) where the terminal substituent was systematically varied. We find that extending the C(5)-C(5a) double bond with unsaturated substituents provides bicyclomycin derivatives with excellent inhibitory activities in the biochemical assay, and that enhanced inhibitory activity is observed for the C(5E) geometrical isomer compared with its C(5Z) counterpart. Finally, C(5a)-substituted dihydrobicyclomycin inhibitory activity appears to be tightly regulated by the nature and spatial placement of the C(5a)-terminal substituent with respect to the [4.2.2]-bicyclic ring system. The observed biochemical activities for the C(5a)-extended conjugated bicyclomycin derivatives and the (5E) and (5Z) isomers were correlated with a structural model for the 1-rho complex.

5a-formylbicyclomycin: studies on the bicyclomycin-Rho interaction.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action is the rho transcription termination factor. A new bicyclomycin irreversible inactivator, 5a-formylbicyclomycin (3), was prepared to provide information concerning the bicyclomycin-rho inactivation process and the drug's binding pocket within rho. The apparent I(50) value for 3 was 35 microM, showing that 3 was a more effective inhibitor of rho poly C-dependent ATPase activity than 1 (I(50) = 60 microM). Mechanistic studies demonstrated that 3 inhibited poly C-dependent ATP hydrolysis, in part, by a reversible, noncompetitive pathway with respect to ATP (K(i) = 62 microM). Incubation of 3 with rho led to efficient imine formation. Adding excess 1 to solutions containing 3 and rho prevented imine formation, demonstrating that 1 and 3 bind to the same active site in the protein. The 3-rho imine was stabilized by either ATP or ADP or by both, and was converted to the nonreversible 3-rho amine adduct upon treatment with NaBH(4). Mass spectrometric analysis of the amine provided a stoichiometry of approximately five bound 3 per rho hexamer indicating the number of bicyclomycin binding sites for the rho hexamer is between five and six. Monomer exchange experiments using modified 3-rho amine and wild type rho demonstrated that no more than two modified subunits per rho hexamer are sufficient to halt poly C-dependent rho ATPase activity.

Rho transcription factor: symmetry and binding of bicyclomycin.

The antibiotic bicyclomycin inhibits rho-dependent termination processes by interfering with RNA translocation by preventing RNA binding at the translocation site or by uncoupling the translocation process from ATP hydrolysis. Previous studies have shown that bicyclomycin binds near the ATP hydrolysis pocket on rho. The hexameric structure of rho indicates that it is in a class of enzymes with strong sequence similarity to F(1)-ATP synthase. The bicyclomycin derivative 5a-formylbicyclomycin, an inhibitor comparable to bicyclomycin, was previously shown to form a stable imine with rho and when reduced to the amine with NaBH(4) to singly label five of the six rho subunits. Lysine-336 was identified by mass spectrometric analysis of trypsin-digested fragments as the site of 5a-formylbicyclomycin adduction. A model of rho was made by threading the rho sequence on the known crystal structure of the alpha and beta subunits of F(1)-ATP synthase. The model, along with information concerning the extent and site of 5a-formylbicyclomycin adduction, indicates an overall C6 symmetry for rho subunit organization. We propose that the sequence similarity between rho and F(1)-ATP synthase extends to a similar quaternary structure and an equivalent enzyme mechanism. The proposed mechanism of RNA translocation coupled with ATP hydrolysis changes the overall symmetry of rho from C6 to C6/C3.

Identifying the bicyclomycin binding domain through biochemical analysis of antibiotic-resistant rho proteins.

Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket.

Evidence for the location of bicyclomycin binding to the Escherichia coli transcription termination factor Rho.

The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174-184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys181. These findings provide the first structural information concerning the site of Bcm binding in Rho.

Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.

Transcription regulation and transcript stability of a light-repressed transcript, lrtA, from the cyanobacterium Synechococcus sp. PCC 7002 were studied using ribonuclease protection assays. The transcript for lrtA was not detected in continuously illuminated cells, yet transcript levels increased when cells were placed in the dark. A lag of 20 to 30 min was seen in the accumulation of this transcript after the cells were placed in the dark. Transcript synthesis continued in the dark for 3 h and the transcript levels remained elevated for at least 7 h. The addition of 10 microM rifampicin to illuminated cells before dark adaptation inhibited the transcription of lrtA in the dark. Upon the addition of rifampicin to 3-h dark-adapted cells, lrtA transcript levels remained constant for 30 min and persisted for 3 h. A 3-h half-life was estimated in the dark, whereas a 4-min half-life was observed in the light. Extensive secondary structure was predicted for this transcript within the 5' untranslated region, which is also present in the 5' untranslated region of lrtA from a different cyanobacterium, Synechocystis sp. PCC 6803. Evidence suggests that lrtA transcript stability is not the result of differences in ribonuclease activity from dark to light. Small amounts of lrtA transcript were detected in illuminated cells upon the addition of 25 microg mL-1 chloramphenicol. The addition of chloramphenicol to dark-adapted cells before illumination allowed detection of the lrtA transcript for longer times in the light relative to controls without chloramphenicol. These results suggest that lrtA mRNA processing in the light is different from that in the dark and that protein synthesis is required for light repression of the lrtA transcript.

Role of the C(6)-hydroxy group in bicyclomycin: synthesis, structure, and chemical, biochemical, and biological properties.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. A recent structure-activity relationship study of 1 showed that replacing the C(6)-hydroxy group with alkoxy and thioalkoxy substituents led to dramatic losses of inhibitory activity in rho biochemical assays. The origin for this structural specificity has been explored by the synthesis and chemical, biochemical, and biological evaluation of C(6)-amino- (13), C(6)-(hydroxylamino)-(14), and C(6)-mercaptobicyclomycin (15). These compounds, like 1, are capable of entering into hydrogen bond donor interactions with rho and are capable of undergoing C(6) ring opening to generate alpha, beta-unsaturated carbonyl, imine, or thione systems. The chemical reactivity of 13-15 was compared with that of 1. We observed that 1, upon treatment with EtSH under moderate and basic conditions, readily underwent C(6) hemiaminal bond cleavage followed by conjugate addition to beta-methylene-alpha-ketoamide 2 to give Michael addition adducts whereas 13-15 reacted by initial cleavage of the C(1)-O(2) bond. Biochemical and biological assays of 13-15 and related analogues demonstrated that the C(6) hydroxy group in 1 was essential for activity. We found that replacing the C(6)-hydroxy group in 1 with weaker hydrogen bond donors led to low inhibitory activities in the rho-dependent ATPase and transcription termination assays. None of the bicyclomycin derivatives exhibited antibiotic activity against E. coli W3350 cells at a 32 mg/mL concentration. The apparent specificity for the C(6)-hydroxy group in 1 suggests that an efficient hydrogen bond donor interaction from the C(6)-hydroxy group to rho or the C(6) hemiaminal bond cleavage to 2 or both is necessary for drug function.

Conserved gene clusters in bacterial genomes provide further support for the primacy of RNA.

Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome.

The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho.

The interaction of Rho and the antibiotic bicyclomycin was probed using in vitro transcription termination reactions, poly(C) binding assays, limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C)10. The approximate I50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon template was 5 microM. At antibiotic concentrations near the I50 value, bicyclomycin inhibition of Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts. Bicyclomycin did not inhibit poly(C) binding to Rho. In the presence of poly(dC), bicyclomycin showed a reversible mixed inhibition of the ribo(C)10-stimulated ATPase activity. The extrapolated Ki for bicyclomycin was 2.8 microM without ribo(C)10 and increased to 26 microM in the presence of ribo(C)10. Correspondingly, the Km(app) for ribo(C)10 without bicyclomycin was 0.8 microM and with bicyclomycin was 5 microM at infinite inhibitor concentration. The data suggested that the antibiotic binds to Rho, influencing the secondary RNA binding (tracking) site on Rho and slows the tracking of Rho toward the bound RNA polymerase.

Bicyclomycin and dihydrobicyclomycin inhibition kinetics of Escherichia coli rho-dependent transcription termination factor ATPase activity.

The primary site of action for the novel antibiotic, bicyclomycin, in Escherichia coli has been identified to be the rho transcription termination factor. The inhibition of rho poly(C)-stimulated hydrolysis of ATP by bicyclomycin has been found to proceed by a non-competitive, reversible pathway with respect to ATP (Ki = 20 microM). Inhibition by dihydrobicyclomycin was similar (Ki = 75 microM). No change in the inhibitory properties of the antibiotic was observed under the assay conditions with the two rho mutants, Cys202Gly and Cys202Ser, indicating that Cys-202 does not affect drug binding to rho. Prolonged incubation (32 degrees C, 12 h) of wild-type rho with bicyclomycin (20 mM) led to protein degradation and a slow, permanent loss of rho ATPase activity after dialysis. Evidence was obtained that trace amounts of proteases present with bicyclomycin were responsible for the observed protein degradation. Treatment of wild-type and mutant rho proteins with purified bicyclomycin (25 mM) led to approximately 80% loss of ATPase activity after dialysis with no apparent loss of protein. However, a reduction of the electrophoretic mobility of the bicyclomycin-treated rho versus wild-type rho was seen. Addition of either ATP or poly(C) to wild-type rho led to partial protection against bicyclomycin inactivation, while inclusion of both ligands provided near complete protection against inactivation. The observed loss of ATPase activity upon prolonged incubation of rho with excess purified bicyclomycin is attributed to the covalent modification of the protein by the antibiotic at multiple sites.

A light-repressed transcript found in Synechococcus PCC 7002 is similar to a chloroplast-specific small subunit ribosomal protein and to a transcription modulator protein associated with sigma 54.

The gene encoding a novel light-repressed transcript (lrtA) contained within a 2.7-kbp EcoRI fragment has been cloned and sequenced from the unicellular cyanobacterium, Synechococcus PCC 7002. Northern analysis indicates that this transcript is synthesized rapidly in the dark, but upon 20 min of illumination, transcript levels fall below detectable limits. An open reading frame was located 378 bases from the start of the transcript which encodes a 21-kDa protein with significant homology to two hitherto different proteins. The protein sequence LrtA showed 37% sequence identity and 58% sequence similarity to the chloroplast-specific small subunit ribosomal protein, S30, and 37% sequence identity and 60% sequence identity and 60% sequence similarity to the reported transcription modulator protein of sigma 54 found in Klebsiella pneumonia and Azotobacter vinelandii. Expression of the lrtA gene product is not detectable within 1 h after placing the cells in the dark, however, within 2.5 min of illumination, [35S]methionine incorporated into a 21-kDa protein. To a lessor extent, [35S]methionine incorporation into a 17- and a 14-kDa protein was also seen which was followed by two other recognizable waves of translation at 5 and 10 min. This incorporation was not blocked by rifampicin added to dark-adapted cells prior to illumination. [35S]Methionine pulsed-labeling experiments suggested that the translation of lrtA occurred only during the first 10 min of reillumination of dark-adapted cells. The loss of initial [35S]methionine labeling in the light of the 21-kDa protein in a kanamycin-interrupted lrtA gene mutant suggests that the lrtA codes for the 21-kDa protein.

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.

Transcription termination factor rho: the site of bicyclomycin inhibition in Escherichia coli.

Bicyclomycin is a novel, commercially important antibiotic. Information concerning the site of bicyclomycin inhibition in Escherichia coli has been obtained by the production of bicyclomycin resistant mutants by UV irradiation. Selection by growth in the presence of bicyclomycin of a plasmid clone library generated from a highly resistant mutant in recipient antibiotic-sensitive host cells (E. coli strain W3350) has led to the characterization of three different plasmids that confer drug resistance, which contained the gene encoding the transcription termination factor, rho. These mutant rho genes contained single base changes at nucleotide positions 656, 796, and 1009. Preliminary mechanistic information has been obtained by monitoring the polyC-dependent ATPase activity of rho in the absence and presence of bicyclomycin and dihydrobicyclomycin. Addition of bicyclomycin to aqueous solutions containing rho and ATP led to a decrease in the release of inorganic phosphate with an I50 value of 60-70 microM bicyclomycin. This inhibition is comparable to the drug concentration needed to inhibit bacterial growth on plates. No loss of activity was observed when a similar concentration of dihydrobicyclomycin was used in place of bicyclomycin, while use of 10-fold higher concentrations of this derivative led to partial rho inhibition. PolyC-dependent ATPase activity from partially purified rho isolated from the mutant BCMr108 was not inhibited by bicyclomycin at concentrations (200 microM) found to completely inhibit wild-type rho. These cumulative findings are consistent with the notion that bicyclomycin expresses its activity by interfering with the polyC-dependent ATPase activity of rho.

Cloning and sequencing of the petBD operon from the cyanobacterium Synechococcus sp. PCC 7002.

The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f.

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108-114, and CPCHGS, residues 128-133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20-24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250-269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.

Light-regulated methylation of chloroplast proteins.

Protein carboxyl methyltransferases, which catalyze transfer of methyl groups from S-adenosyl-L-methionine to the free carboxyl groups of acidic amino acids in proteins, can be divided into two classes based on several characteristics, such as the stoichiometry of substrate protein methylation, base stability of the incorporated methyl group, specificity for substrate, and participation in a regulatory system with which methylesterases are associated. The presence of such an enzyme in a photosynthetic system was demonstrated in the present work. The extent of methylation of chloroplast proteins was stimulated 30% by light and then decreased by the same amount in the presence of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1', 1'-dimethylurea or uncouplers of phosphorylation, indicating a dependence of the methyltransferase activity on photosynthetic electron transport and the trans-membrane delta pH. The light-independent, as well as the light-dependent, activity is probably of chloroplast origin since the extent of light stimulation in the purified thylakoid membranes and the stromal fraction was similar, and at low concentrations of S-adenosyl-L-methionine the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was found to be the predominant substrate. The labeling pattern of chloroplast proteins and labeling of an exogenous nonchloroplast protein indicated that the methyltransferase activity was not substrate-specific, although at low concentrations of the methyl donor, the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was labeled almost exclusively. Based on the low stoichiometry (less than 100 pmol/mg protein) of the methylation, its base lability, irreversibility, and the lack of substrate specificity except at very low concentrations of methyl donor, it was inferred that the chloroplast methyltransferase is best classified as a class II system that may function as part of a repair mechanism to replace racemized amino acids.

Large-scale purification of active cytochrome b6/f complex from spinach chloroplasts.

A preparation is described through which large quantities of pure, active cytochrome b6/f complex can be isolated from spinach chloroplasts. The resulting complex is at least 90% pure with respect to the maximum content of redox centers, consists of four polypeptides according to polyacrylamide gel electrophoresis, and lacks both ferredoxin: NADP+ oxidoreductase and the high molecular weight form of cytochrome f seen in some other preparations. The complex contains 2 mol b6 and 2 atoms of nonheme iron per mole of cytochrome f, and possesses a high plastoquinol-plastocyanin oxidoreductase activity (Cyt f turnover no. 20-35 s-1). The present preparation should be helpful in the effort to crystallize the cytochrome b6/f complex.