Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Diacylglycerol-transporting lipoproteins and flight in Locusta

Evidence from chromatographic and heparin precipitation studies shows that the ‘heparin-soluble’ lipoprotein, A⁺, forms in the haemolymph during flight. In locusts flown continuously for 60 min, lipoprotein A⁺ occurs in the haemolymph at low concentrations but accumulates during a short rest period following flight. After injections of tissue extracts containing adipokinetic hormone (AKH), A⁺ accumulates in the haemolymph but disappears more rapidly in flying locusts than in resting locusts. This difference in the rate of disappearance of diacylglycerol from the lipoprotein A⁺ can be used to estimate its rate of utilization during sustained flight (approx. 100μg. min^?1 from 45–90 min of flight). It is suggested that lipoprotein A⁺ is the major carrier of diacylglycerol from the fat body to the flight muscles during prolonged flight. The steady state concentrations of total diacylglycerol and ‘heparin-soluble’ diacylglycerol during continuous flight are unaffected when tissue extracts containing AKH are injected before flight. This suggests that there is a close homeostatic control over the steady state concentration of haemolymph lipid during flight.     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

Juvenile hormone-induced biosynthesis of vitellogenin in Leucophaea maderae

Isolated abdomens of virgin female Leucophaea maderae, a South American cockroach, could be induced by a single injection of juvenile hormone I to synthesize vitellogenin. Synthetic capacity was assayed by removal of the fat body at various times after induction and incubation in organ culture with radioactive leucine for 5 h. Under these conditions, active tissues secrete radioactive vitellogenin into the medium after a lag of 2 h and continue secretion at a fixed rate for up to 8 h. Vitellogenin in the tissue reaches a steady-state level after about 3 h. By 5 h, approximately 80% of the newly synthesized vitellogenin can be found in the medium and constitutes 75% of the secreted protein.Vitellogenin does not appear in the medium until 18 h after juvenile hormone induction, rises to a maximum between 72 and 96 h, and has declined to half by 120 h postinduction. At the maximum, about 75% of the total protein secreted is vitellogenin, a more than 50-fold stimulation over injected controls. Since virtually the same quantitative effect of juvenile hormone was found in abdomens with and without ovaries, secondary stimulation by ovarian hormones does not appear to be involved in vitellogenin synthesis in this insect.Oocyte vitellogenin exists as a 560,000 molecular weight monomer complex and a 1,590,000 molecular weight trimer. The smaller complex is only found in young oocytes. By sodium dodecyl sulfate-gel electrophoresis, we find that the 560,000 molecular weight 14S monomer consists of four discrete peptides in the ratio A₁B₁C₂D₂ with molecular weights 118,000, 87,000, 57,500, and 96,000, respectively. The 28S trimer contains only the three peptides A, B, and C in the ratio A₁B₃C₂. These stoichiometries satisfactorily account for the molecular weights of both complexes when corrected for the lipid content. Maturation of 14S to 28S probably involves specific proteolytic conversion of D to B^D, a different peptide of the same size as B, suggesting that the true composition of 28S is A₁B₁B₂^DC₂.The 14S vitellogenin synthesized and secreted by fat body in tissue culture consists mostly of larger polypeptides. There is a major fraction of 179,000 molecular weight, a minor fraction of >260,000 molecular weight, and other smaller polypeptides. Although the exact relationship between these polypeptides and the mature subunits of vitellogenin are not yet clear, it is evident that the multiple protein subunits of this complex protein are synthesized as one or more precursor proteins, followed by proteolytic processing to the mature size.     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.     

Distribution of antigens in established cell lines of Drosophila melanogaster

Antibodies prepared against two Drosophila cell lines together with antibodies prepared against other Drosophila extracts were used to study the distribution of antigens in 10 cell lines. The results suggest: (1) that cultured cells express differentiated functions; (2) that each cell line, including sub-clones of a clone, is unique; and (3) that the cell lines are derived from the lateral ectoderm.     

The effects of allatectomy and ovariectomy on haemolymph proteins and lipid mobilization in Locusta

After ovariectomy the concentrations of diacylglycerol and protein in the haemolymph increase markedly. The increased diacylglycerol is associated with increased quantities of the ‘heparin-precipitable’ protein (lipoprotein A) that carries diacylglycerol in the blood of normal resting locusts. After the injection of adipokinetic hormone (AKH), the blood of ovariectomized locusts contains only slight quantities of the ‘heparin-soluble’ lipoprotein A⁺ whereas this forms in large amounts in the blood of sham-operated locusts after AKH injection. After allatectomy, the increase in the adipokinetic response is slower and the full level of responsiveness observed in sham-operated locusts is never attained. Nevertheless, allatectomized locusts develop a marked adipokinetic response which tends to stabilize as they age; it does not deteriorate as it does in aged sham-operated locusts.The effects of ovariectomy on blood metabolites can be prevented completely by allatectomy, but only partially by cautery of the cerebral neurosecretory cells. Treatment with a juvenile hormone analogue (JHA R-20458) counteracts the effects of allatectomy in ovariectomized locusts.     

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

Comparative studies of the structure and composition of the plasmalemma and the tonoplast in Saccharomyces cerevisiae

The two membranes, plasmalemma and tonoplast (Saccharomyces cerevisiae H 1022), are characterized ultrastructurally by their different texture in the corresponding freeze-fracture faces and their silver staining properties.Biochemical characterization with regard to proteins and lipids indicated that the ratio of protein to lipid is significantly higher in the plasmalemma as compared to the tonoplast. Moreover, a pronounced difference appears to exist for both the amount and the composition of total lipids, phospholipids and sterols. The protein patterns of the plasmalemma and the tonoplast reveal only minor differences, as judged by sodium dodecyl sulphate gel electrophoresis.     

Primary structure of the Klebsiella serotype 16 capsular polysaccharide

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α-D-Glcp-(1/ar4)β-D-GlcAp-(1/ar4)-α-L-Fucp-(1/ar chain with a β-D-Galp-(1→ branch at position 4 of the D-glucosyl residue.