Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

Variation in flavonoid patterns in relation to chromosome changes in Gibasis schiedeana

The distribution of $\text{C-}\text{glycoflavones}$ has been examined in the leaves of plants from five diploid and 12 tetraploid populations of Gibasis schiedeana. Five different chromatographic patterns were found; one occurs only in a single population of diploid plants, two were found in seven populations of tetraploids and the other two in the remaining plants from both diploid and tetraploid populations. Flavonoids were also investigated in three different types of artificially produced triploids. The effects of the changes in ploidy, which are accompanied by Robertsonian fusion and variation in the number of B chromosomes as contributory factors in the observed differences in flavonoid patterns are discussed.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Linear oligopeptides. 56. Critical size for development of β-and α-helical structures of monodispersed homo-l-oligomethionines in the solid state

The infrared absorption properties of monodispersed, chemically and optically pure ‘amino-PEG’-bound linear homooligopeptides having the general formula $\text{t-}\text{Bx(}\text{l}\text{-Met)}{}_{\mathrm{n}}\text{NHPEG}$, n = 1–15 and H₂⁺(l-Met)_nNHPEG, n = 1–14 have been investigated in the solid state. The critical sizes for development of the β-structure and α-helix (n = 3 and n = 13, respectively), and the effect, on the latter, of the solvent from which the solid samples are cast, have been established.     

Induction of fertile oestrus in seasonally anoestrous ewes with low doses of Gn-RH

Oestrus, conception and lambing performance were assessed in progesterone-primed seasonally anoestrous ewes induced to ovulate with gonadotrophin-releasing hormone (Gn-RH), which was administered intravenously for 48 h as either injections of 250 ng at 2-h intervals (n = 15) or as a continuous infusion at the rate of 125 ng/h (n = 12) or 250 ng/h (n = 12).In $\text{14}\text{15}$ of the ewes injected with Gn-RH, a preovulatory LH peak was recorded at a mean time interval of 33.9 ± 1.8 h after the start of treatment. All ewes displayed oestrus and all ovulated, with a mean ovulation rate of 1.67 ± 0.13. Eleven ewes were diagnosed as pregnant and subsequently lambed. Following infusion of Gn-RH, preovulatory LH peaks were recorded in $\text{21}\text{24}$ ewes at a mean time of 36.1 ± 2.9 h (125 ng/h) and 34.7 ± 2.0 h (250 ng/h). All but two of the ewes displayed oestrus and $\text{23}\text{24}$ ovulated. The group mean ovulation rates of 1.27 ± 0.14 (125 ng/h) and 1.75 ± 0.22 (250 ng/h) were not significantly different. Eleven of the 22 ewes mated were diagnosed as pregnant and produced live lambs.These results suggest that fertility of Gn-RH-induced ovulations in seasonally anoestrous ewes is comparable to that apparent in ewes ovulating spontaneously during the breeding season.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

Inhibitory beta-adrenoceptors in the urinary bladder of the rat

The β-adrenoceptors of the urinary bladder were investigated in the rat $\text{in}$$\text{vivo}$. Isoprenaline, and the β₂-stimulating agents terbutaline and salbutamol elicited relaxation of the detrusor muscle decreasing the intravesical pressure. The responses were not affected by the β₁-blocking agents practolol or H 93/26 but were totally abolished by propranolol and the β₂-blocking agent H $\text{35}\text{25}$. Noradrenaline given after dihydro-ergotamine caused relaxation of the detrusor muscle and this response was completely blocked by propranolol and H $\text{35}\text{25}$. It is concluded that the β-adrenoceptors of the rat urinary bladder belong to the type of inhibitory receptors classified as β₂-receptors in other organs.     

Reproductive studies of Brahman cattle I. Behavioral effect of various dose levels of Estradiol-17β upon ovariectomized Brahman,Brahman × Hereford and Hereford cows

Six ovariectomized mature cows each of Brahman (B), Brahman × Hereford (BH) or Hereford (H) breeding were injected intramuscularly with Estradiol-17β (E). Dose levels of 1, 2, 4 and 8 mg E were given in 2 ml corn oil. Cows were allowed a 2 week recovery period between treatments. After injection the cows were placed with 18 epididymectomized bulls and observed constantly for 36 hours. B failed to accept the bulls at any E dose level. Proportions of BH accepting the bull were $\text{2}\text{6}$, $\text{6}\text{6}$, $\text{6}\text{6}$ and $\text{6}\text{6}$ and proportions of H accepting the bull were $\text{5}\text{6}$, $\text{6}\text{6}$, $\text{5}\text{6}$ and $\text{6}\text{6}$ at 1, 2, 4 and 8 mg E, respectively. BH were less responsive at 1 mg E than H (P<.10) and B were less responsive at any level (P<.005). The number of stances increased significantly with dose level (P<.005) and a breed × dose level interaction (P<.10) was found. The duration of standing estrus behavior was longer in H cows at 1 and 2 mg E than in BH (P<.05) but was identical at 4 and 8 mg E. Duration of estrus was shorter in B except at the 2 mg dose level. Breed (P<.005) and breed × dose level interactions (P<.05) were found. Response time (injection of E to stance event) did not differ between dosages of E within breed groups. However, response time was significantly longer in B (19.3 hrs, P<.05) versus the response time of either H (10.1 hrs) or BH (12.8 hrs). If homosexual stance behavior is accepted as estrus, B were less responsive at 1, 2, 4 and 8 mg E than were BH or H (P<.10).     

The effect of redox potential on the kinetics of fluorescence induction in Photosystem II particles from Phormidium laminosum. Sigmoidicity,energy transfer and the slow phase

Fluorescence induction curves in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited Photosystem (PS) II particles isolated from the blue-green alga Phormidium laminosum have been analysed as a function of redox potential. Redox titration of the initial fluorescence indicated a single component with E_m,7.5 = +30 mV (n = 1) (Bowes, J., Horton, P. and Bendall, D.S. (1981) FEBS Lett. 135, 261–264). Despite this simplified electron acceptor system and the small number of chlorophylls per reaction centre, a sigmoidal induction curve was nevertheless seen. Sigmoidicity decreased as Q was reduced potentiometrically prior to induction such that the induction was exponential when the ratio $\text{F}{}_{\mathrm{<mtext>i</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}\text{= 0.64}$. These particles also showed a slow (β) phase of induction which titrated with an E_m value slightly more positive than that of the major quencher. It is concluded that the sigmoidal shape of the fluorescence induction curve observed in Phormidium PS II particles is not a consequence of a requirement for two photons to close the PS II reaction centre, but is generated as a result of energy transfer between photosynthetic units comprising one reaction centre per approx. 50 chlorophylls. Also, the existence of PS II heterogeneity (PS II_α, PS II_β centres) does not require a structurally differentiated chloroplast, but may only indicate the extent of aggregation of PS II centres.     

Crystallographic study of the anti-tumor protein ricin

The anti-tumor protein ricin (also called RCA_II) has been crystallized in a form suitable for X-ray diffraction analysis. The crystal symmetry is orthorhombic spacegroup P2₁2₁2₁ with $\text{a = 72.9, b = 79.1, c = 114.7}\text{A}\text{?}$ and a single ricin molecule of molecular weight 65,000 per asymmetric unit. The crystals diffract well and are stable in the X-ray beam. At least one useful heavymetal derivative has been found as part of a high-resolution study.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β-p-bromobenzoyl ester of diol B was prepared from 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; $\text{a = 9.24}\text{A}\text{?}$, $\text{b = 12.61}\text{A}\text{?}$, $\text{c = 7.03}\text{A}\text{?}$, α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.     

The leaf flavonoids of the Zingiberales

A survey of flavonoids in the leaves of 81 species of the Zingiberales showed that, while most of the major classes of flavonoid are represented in the order, only two families, the Zingiberaceae and Marantaceae are rich in these constituents. In the Musaceae (in 9 species), Strelitziaceae (in 8 species) and Cannaceae (1 of 2 species) flavonol glycosides were detected in small amount and in the Lowiaceae no flavonoids were fully identified. In the Zingiberaceae kaempferol (in 22%), quercetin (72%) and proanthocyanidins (71%) are distributed throughout the family. The two subfamilies of the Zingiberaceae may be distinguished by the presence of myricetin (in 26%), isorhamnetin (10%) and syringetin (3%) in the Zingiberoideae and of flavone $\text{C-}\text{glycosides}$ (in 86% of taxa) in the Costoideae. A number of genera have distinctive flavonol profiles: e.g. Hedychium species have myricetin and quercetin. Roscoea species isorhamnetin and quercetin and Alpinia species kaempferol and quercetin glycosides. A new glycoside, syringetin 3-rhamnoside was identified in Hedychium stenopetalum. In the Zingiberoideae flavonols were found in glycosidic combination with glucuronic acid, rhamnose and glucose but glucuronides were not detected in the Costoideae or elsewhere in the Zingiberales. The Marantaceae is chemically the most diverse group and may be distinguished from other members of the Zingiberales by the occurrence of both flavone $\text{O-}$ and $\text{C-}\text{glycosides}$ and the absence of kaempferol and isorhamnetin glycosides. The distribution of flavonoid constituents within the Marantaceae does not closely follow the existing tribai or generic limits. Flavonols (in 50% of species). flavones (20%) and flavone $\text{C-}\text{glycosides}$ (40%) are found with similar frequency in the two tribes and in the genera Calathea and Maranta both flavone and flavonol glycosides occur. Apigenin- and luteolin-7-sulphates and luteolin-7,3′-disulphate were identified in Maranta bicolor and M. leuconeura var. kerchoveana and several flavone $\text{C-}\text{glycosides}$ sulphates in Stromanthe sanguinea. Anthocyanins were identified in those species with pigmented leaves or stems and a common pattern based on cyanidin-and delphinidin-3-rutinosides was observed throughout the group. Finally the possible relationship of the Zingiberales to the Commelinales, Liliales, Bromeliales and Fluviales is discussed.     

Midpoint potential of signal II (slow) in Tris-washed photosystem-II particles

In Tris-washed Photosystem-II particles we are able to induce an EPR signal in the dark by addition of an iridium salt (K₂IrCl₆). This signal is attributed to signal II_s (slow) (D⁺) and the redox titration gives an E_m value of 760 mV for the couple $\text{D}{}^{\mathrm{+}}\text{D}$. On the basis of our previous studies on the equilibrium between D⁺Z and DZ⁺ (K = 10⁴) (Boussac, A. and Etienne, A.L. (1982) Biochem. Biophys. Res. Commun. 109, 1200–1205), we therefore attribute a value of 1 V for the E_m of the $\text{Z}{}^{\mathrm{+}}\text{Z}$ couple. A second effect of K₂IrCl₆ is to modify the spectral characteristics of signal II. We conclude that K₂IrCl₆ is able to change the environment of the species from which signal II_s and signal II_f originate.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

Natural intergeneric hybrids betweenAster ageratoides subsp.ovatus (2n=36) andKalimeris incisa (2n=72) were found. All of the hybrids studied were found to have 2n=72, 18 more chromosomes than a regular F₁ hybrid. The hybrids were found to be of two types: one having 18 large chromosomes ofovatus, and the other having 9 large chromosomes of the same subspecies. In meiosis of the PMCs of the hybrid with 18 large chromosomes, a regular chromosome configuration, 36_II, was observed. In PMCs of the hybrid with 9 large chromosomes an irregularity of chromosome pairings was observed, showing varied chromosome configurations: 35_II+2_I, 34_II+4_I, 33_II+6_I, I_III+33_II+3_I, 1_IV+32_II+4_I, 32_II+8_I, 31_II+10_I, 29_II+14_I, 3_III+29_II+5_I. Most univalents were large, but a few were small. The hybrids with 18 large chromosomes were found to be partial amphidiploid and possessing double chromosome complements ofovatus. The hybrids with 9 large chromosomes were found to be the first backcrossed generation between the hybrid with 18 large chromosomes andK. incisa.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

ADP-ribosylation of nuclear protein A24

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) $\text{Proc}$. $\text{Natl}$. $\text{Acad}$. $\text{Sci}$. $\text{USA}$$\text{74}$, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.