Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Identification of a protein component of the antimycin-binding site of the respiratory chain by photoaffinity labeling

Deformamido azido antimycin A (DAA)³ was synthesized and was found to resemble its parent compound, antimycin A, in its inhibitory effects on Complex III, with the exception that its effects were freely reversible with dilution. However, exposure to ultraviolet light converted the effects of DAA from reversible to irreversible, indicating formation of a covalent linkage between DAA and the antimycin site of Complex III.After treatment of Complex III with tritium-labeled DAA followed by gel electrophoresis in a solvent system of sodium dodecyl sulfate (SDS), DAA ³H was found to be associated with a single protein component distinct from the previously characterized components of the complex. The molecular weight of the DAA-labeled protein is approximately 11,500 g-mol^?1 as estimated from its electrophoretic mobility in the SDS-polyacrylamide gel system.     

Further studies on the binding of DCCD to cytochromeB and subunit VIII of complex III isolated from beef heart mitochondria

Complex III (the cytochromeb-c ₁ complex) from beef heart mitochondria was incubated with [¹⁴C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochromeb) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [¹⁴C]DCCD resulted in a 50% decrease in the binding of DCCD to both chtochromeb and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochromeb and subunit VIII of complex III from beef heart mitochondria and suggest that cytochromeb may play a role in proton translocation.     

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA₁, GA₄ and GA₄-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA₈ and GA₃₄ did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA = 5-azido-7-[³H]indole-3-acetic acid - azido NPA = 5-azido-[3,6-³H]1-N-napthylpthalamic acid - BTP = 1,3-bis(Tris(hydroxymethyl)methylamino)-propane - GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate - [¹²⁵I]GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate - NPA = 1-Naphthylphthalmic acid - PAGE = Polyacrylamide gel electrophoresis - PMSF = phenylmethylsulfonyl fluoride - SDS = Sodium dodecyl sulphate - TLCK = L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μm) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μm-succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μm; 4 μm-antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μm) and antimycin A (4 μm) by 110%. Azide (1 mm) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 mm-KCN (IC₅₀ = 0.8 mm). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

Cholinergic Binding to the Receptor Proteolipid from Torpedo Electroplax Separated by Ion Exchange Chromatography

Abstract

Proteolipids (i.e., hydrophobic proteins) have been extracted with chloroform-methanol (2:1) from lyophilized Torpedo electroplax, and fractionated on a DEAE-cellulose column. The elution system consisted of the same solvent and a gradient of the hydrophobic ion ptoluene sulfonate (0.1–100mM). The three fractions obtained (I, II and III) have different content of protein, lipid P, and reducing sugars. The amino acid composition shows a higher proportion of hydrophobic residues in I and more charged ones in fractions II and III. Polyacryl amide gel electrophoresis of fraction I shows a single major band of 39 kdaltons; in fractions II and III a major band of 42 kdaltons and fainter bands in the range 62–68 kdaltons are observed.

Fraction I has the highest specific binding for [³H]-acetylcholine (7.1 nmol/mg protein) and [³H]α-bungarotoxin (5.2 nmol/mg protein). The nicotinic nature of this proteolipid was demonstrated by blocking experiments. The Scatchard plot showed two affinity sites for [³H]-acetylcholine (Kd₁ = 3nM and Kd₂ = 1.1 μm). 4-(N-maleimido) pheny1 [³H]trimethylammonium specifically labeled the 39 kdaltons band.

The possible factors involved in the fractionation of proteolipids are discussed. The findings suggest that the 39 kdaltons polypeptide contains the receptor site for acetylcholine and that the other proteolipid components may play a different function in the membrane.     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Gibberellin metabolism in excised lettuce hypocotyls: Response to GA9 and the conversion of [3H]GA9

Elongation growth and gibberellin (GA₉) metabolism in excised hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Exogenously supplied GA₉ stimulates elongation of hypocotyl sections and this response is intermediate between that elicited by GA₁ or GA₂₀ and GA_4/7 mixture. Although uptake of radioactivity from [³H]GA₉ increases with time, this gibberellin does not accumulate in the tissue but is rapidly converted to a compound with HPLC properties resembling those of [³H]GA₂₀. After 2 h incubation in [³H]GA₉, the presumptive GA₂₀ represents 90% of the acidic ethyl acetate-soluble radioactivity in the tissue. Radioactivity is also associated with an acidic butanol-soluble fraction containing two components resolvable by HVE. The major component is similar in electrophoretic properties to a GA-glucosyl ether while the other compares to a GA-glucosyl ester. Conversion of [³H]GA₉ to its [³H]GA₂₀-like metabolite is reduced by addition of carrier GA₉ or GA_4/7 at concentrations as low as 1 M, while GA₁, GA₃ and L-proline are without effect. Formation of the GA₂₀-like compound can be blocked by the addition of 2,2-dipyridyl, and this inhibitory effect of dipyridyl can be reversed by addition of Fe²⁺. At 200 M dipyridyl, elongation growth as well as [³H]GA₉ metabolism are reduced by 80%. The relationship of the metabolism of GA₉ to the growth response is discussed.Abbreviations AB butanol-soluble - AE ethyl-acetate-soluble - GA gibberellin - GA₁, GA₄ gibberellin A₁, gibberellin A₄, etc. - TLC thin layer chromatography - HPLC high performance liquid chromatography - HVE high voltage electrophoresis     

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH₂-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-³H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.     

Antimycin-resistant alternate electron pathway to plastocyanin in bovine-heart Complex III

Bovine-heart Complex III can catalyze the reduction of spinach plastocyanin by a decyl analog of ubiquinol-2 at a rate comparable with the rate of plastocyanin reduction by plastoquinol as catalyzed by the cytochromeb ₆–f complex purified from spinach leaves. This plastocyanin reduction as catalyzed by Complex III was almost completely inhibited by myxothiazol at stoichiometric concentrations, partially inhibited by UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) and funiculosin, and was relatively insensitive to antimycin and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). Cytochromec reduction as catalyzed by Complex III displayed a residual, inhibitor-insensitive rate of 5% of the uninhibited rate for each of the three inhibitors, antimycin, myxothiazol, and UHDBT. However, the residual rate that was insensitive to each of the inhibitors added singly was inhibited further by addition of the remaining two inhibitors. From these results it is concluded that plastocyanin reduction involves an electron-transfer pathway through Complex III that is distinct from the pathway utilized for reduction of cytochromec.A portion of the data in this report was presented at the IV International Symposium on Coenzyme Q₁₀, Munich, F.R.G., November, 6–9, 1983.     

Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h^-1). [³H]Gibberellin A₁ ([³H]GA₁) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [³H]gibberellin A₈ ([³H]GA₈) and a conjugate of [³H]GA₈, whereas leaves of dwarf seedlings metabolised [³H]GA₁ more slowly. Roots of both genotypes produced [³H]GA₈-like material at similar rates. Isotopic dilution studies indicated a reduced 2-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12–15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

Metabolism of [1,2-3H]gibberellin A4 by epicotyls and cell-free preparations from Phaseolus coccineus L. seedlings

Cell-free systems were prepared from germinating seed and seedlings of Phaseolus coccineus. Gibberellin A₄ (GA₄)-metabolising activity was detected in vitro using preparations from roots, shoots and cotyledons of germinating seed, but only up to 24 h after imbibition. Cell-free preparations from cotyledons converted [³H]GA₄ to GA₁, GA₃₄, GA₄-glucosyl ester and a putative O-glucoside of GA₃₄, and, in addition converted [³H]GA₁ to GA₈. Preparations from embryo tissues contained 2-hydroxylase activity, converting [³H]GA₄ to GA₃₄ and [³H]GA₁ to GA₈.The presence of GA-metabolising enzymes was also indicated by in-vivo feeds of [³H]GA₄ to epicotyls of intact 4-d-old seedlings, which resulted in the accumulation of GA₁, GA₈, GA₃-3-O-glucoside, GA₄-glucosyl ester, GA₈-2-O-glucoside and a putative O-glucoside of GA₃₄. Gibberellin A₁ was the first metabolite detected, 15 min after application of [³H]GA₄, but after 24 h most of the label was associated with GA₈-2-O-glucoside. Over 90% of the recovered radioactivity was found in the shoot. Within the shoot, movement was preferentially acropetal, and was not dependent upon metabolism of the applied [³H]GA₄.Abbreviations DEAE diethylaminoethyl - GA_n gibberellin A_n - GPC gel permeation chromatography - HPLC-RC high performance liquid chromatography-radio counting - S-1 1000·g supernatant - UDP uridine 5-diphosphate     

On the biosynthesis of 5-hydroxybenzimidazolylcobamide (vitamin B12-factor III) in Methanosarcina barkeri

Exogenous 5-hydroxy-[2-¹⁴C]benzimidazole was transformed by Methanosarcina barkeri into 5-hydroxy-[2-¹⁴C]benzimidazolylcobamide. Thereby the endogenous biosynthesis of 5-hydroxybenzimidazole was completely blocked.Benzimidazole and 5,6-dimethylbenzimidazole were used by M. barkeri to form benzimidazolylcobamide respectively 5,6-dimethylbenzimidazolylcobamide (vitamin B₁₂), but in these cases the endogenous biosynthesis of factor III was not completely suppressed.With [2-¹⁴C]benzimidazole it was demonstrated that this base as well as the benzimidazolylcobamide formed thereof are no precursors in the biosynthesis of 5-hydroxybenzimidazolylcobamide.Glycine instead was found to be a building block for the biosynthesis of 5-hydroxybenzimidazole, since radioactivity from [1-¹⁴C] and [2-¹⁴C]glycine was incorporated, into the base moiety of factor III, but not into its corrin moiety. With [1-¹³C]glycine and ¹³C-NMR-spectroscopy it was shown that C-1 of glycine gets C-3a of 5-hydroxybenzimidazole.[1-¹³C]glycine also led to a single prominent signal in the ¹³C-NMR-spectrum of coenzyme F₄₂₀, this was assigned to C-10a.Thus C-1 of glycine was incorporated into the hydroxybenzene part of 5-hydroxybenzimidazole, whereas it was not incorporated into this part of coenzyme F₄₂₀, indicating that the hydroxybenzene part of these two compounds is not formed from a common intermediate. L-[U-¹⁴C]glutamate led to the exclusive labeling of the corrin ring of factor III, showing that the corrin precursor 5-aminolevulinic acid is formed by the C-5 pathway in M. barkeri.These experiments indicate that the biosynthesis of factor III in the archaebacterium M. barkeri is similar to the corrinoid biosynthesis in the anaerobic eubacteria Eubacterium limosum, Clostridium barkeri, and Clostridium thermoaceticum.     

In-vivo formation of xyloglucan nonasaccharide: A possible biologically active cell-wall fragment

The in-vivo formation of a specific nonasaccharide of xyloglucan was investigated. This nonasaccharide has been reported to have biological activity, inhibiting auxin-induced growth in pea stem segments. Cell-suspension cultures of spinach were grown in the presence of [³H]arabinose and [³H]fucose, and the culture-filtrates were examined for oligosaccharides by gelpermeation chromatography and by paper chromatography. Sixteen [³H]pentose-containing oligosaccharides were found, including twelve that contained the sequence [³H]xylosyl-(16)-glucose, which is diagnostic of xyloglucan. In addition, [³H]fucose-containing oligosaccharides of at least three sizes were found. Radiochemical evidence is presented that one of these oligosaccharides was labelled with both [³H]fucose and with [³H]pentose, and was identical with the major xyloglucan-derived nonasaccharide associated with anti-auxin activity. It was largely present in the form of acylated (possibly acetylated) derivatives. It accumulated extracellularly to a steady-state concentration of about 4.3·10^-7M. This is the first report of the production of a biologically-active oligosaccharide by living plant cells.Abbreviations BAB butanone/acetic acid/H₃BO₃-saturated water (9:1:1) - BAW butan-1-ol/acetic acid/water (12:3:5) - BPW butan-1-ol/pyridine/water/(4:3:4) - DP degree of polymerisation - FAW ethyl acetate/acetic acid/water (10:5:6) - EPW ethyl acetate/pyridine/water (8:2:1) - k _av elution volume relative to Blue Dextran (k _av.=0.0) and glucose (k _av.=1.0) - XG7 XG9 minus the fucose and galactose residues - XG9 the particular xyloglucan nonasaccharide illustrated in Fig. 1 - W water-saturated phenol     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol