Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

The expression of I-Ed molecules in F1 hybrid mice detected with antigen-specific,I-Ed-restricted cloned T-cell lines

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E chain. We have previously analyzed the expression of E ^k and E ^b chains in F₁ hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E expression. In the present study, we have extended our analysis to E ^d expression in F₁ hybrid mice. We show that E ^d is relatively poorly expressed in three F₁ combinations: H-2 ^d× H-2 ^b, H-2 ^d× H-2 ^s, and H-2 ^d× H-2 ^u. The former two crosses express E chains from the H-2 ^dparent only; when recombinant strains carrying E ^b or E ^s and an active E gene are used, E ^d expression is significantly increased. On the other hand, H-2 ^umice synthesize E chains; the poor expression of E ^d chains in this F₁ hybrid apparently reflects the strong preferential association of E ^u chains with all E molecules thus far analyzed. These results confirm that E chains compete for binding to E chains and that preferential association of different allelic forms of E chains with E chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Hormonal regulation of hepatic glycogenolysis inAmphibolurus nuchalis,the western netted dragon: an in vitro study

Summary In mammals hepatic glycogenolysis is controlled by several hormones using cyclicAMP, Ca²⁺ and/or diacylglycerol as intracellular messengers. In contrast, in teleost fish, lungfish and amphibians fewer hormones promote hepatic glycogenolysis, and cyclicAMP is the sole intra-cellular messenger. This suggests that the -adrenergic mechanism became associated with the liver after amphibians separated from the vertebrate line. Reptiles separated later, and the aim of this study is to elucidate the hormonal control of hepatic glycogenolysis in a reptile,Amphibolurus nuchalis, and especially to determine which adrenergic receptor system is operative.InA. nuchalis liver pieces cultured in vitro, adrenaline and glucagon stimulated glycogen breakdown and glucose release, glycogen phosphorylase activity and accumulation of cyclicAMP in the tissue. Neurohypophysial peptides did not affect these parameters. These actions of adrenaline were completely blocked by the -adrenergic antagonist, propranolol and slightly reduced by the -adrenergic antagonist, phentolamine. Removal of Ca²⁺ from the medium and addition of the Ca²⁺ chelator, EGTA, did not block the actions of adrenaline, and the Ca²⁺ ionophore A23187 did not mimic these actions.The -adrenegic ligand [¹²⁵I]-iodocyanopindolol (ICP) bound specifically to an isolated membrane preparation fromA. nuchalis liver with a calculated K_D of 100 pM and a Bmax of 37.6 fmol·mg protein^–1. The adrenergic ligands propranolol, isoprenaline, adrenaline, noradrenaline, phenylephrine and phentolamine displaced ICP with K_D's of 20 nM, 1 M, 4.5 M, 32 M, 35 M and 500 M, respectively. The ₂-adrenergic ligand yohimbine did not bind specifically to the membrane, but at 1 nM and 100 pM, specific binding of the ₁-adrenergic ligand prazosin was 45% of total with a mean of 11.3 fmoles·mg protein^–1 specifically bound.These findings indicate that the glycogenolytic actions of adrenaline are mediated primarily via -adrenergic receptors inA. nuchalis, but that -adrenergic receptors may also play some role in the control of hepatic metabolism.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Voltage dependence of the basolateral membrane conductance in theAmphiuma collecting tubule

Summary The basolateral potassium conductance of cells of most epithelial cells plays an important role in the transcellular sodium transport inasmuch as the large negative equilibrium potential of potassium across this membrane contributes to the electrical driving force for Na⁺ across the apical membrane. In the present study, we have attempted to establish, theI-V curve of the basolateral membrane of theAmphiuma collecting tubule, a membrane shown to be K⁺ selective. TransepithelialI-V curves were obtained in short, isolated perfused collecting tubule segments. The shunt conductance was determined using amiloride to block the apical membrane Na⁺ conductance. In symmetrical solutions, the shuntI-V curve was linear (conductance: 2.2±0.3 mS·cm^–2). Transcellular current was calculated by subtracting the shunt current from the transepithelial current in the absence of amiloride. Using intracellular microelectrodes, it was then possible to measure the basolateral membrane potential simultaneously with the transcellular current. The basolateral conductance was found to be voltage dependent, being activated by hyperpolarization: conductance values at –30 and –80 mV were 3.6±1.0 and 6.6±1.0 mS·cm^–2, respectively. BasolateralI-V curves were thus clearly different from that predicted by the constant field model. These results indicate that the K⁺-selective basolateral conductance of an amphibian collecting tubule shows inward (anomalous) rectification. Considering the electrogenic nature basolateral Na–K-pump, this may account for coupling between pump-generated potential and basolateral K⁺ conductance.     

Enhancement of protein precipitate strength and density by low-frequency conditioning

The use of a continuous, low-frequency conditioning process to alter the structure of protein precipitate aggregates is examined. An increase in the density of aggregates is correlated with the levels of fluid acceleration and hence hydrodynamic stress to which the aggregates are exposed during conditioning. A combination of low-frequency conditioning followed by shear break-up (as in the feed zone to a high-speed disk-stack centrifuge) is shown to result in a precipitate suspension of increased particle size at the fine end of the distribution, and having a greater sedimentation velocity. The resistance of large aggregates to shear disruption is increased by low-frequency conditioning.List of Symbols CR conditioning ratio - CRS conditioning ratio after shearing - d m amplitude of displacement - D m particle size - D _c m critical size for centrifuge recovery - f s^–1 frequency of vibration - G s^–1 mean velocity gradient - Q m³/s volumetric throughput - SR shear ratio - t s ageing time Greek Symbols s^–1 mass-average shear rate - K sedimentation shape factor - _a kg/m³ aggregate density - _f kg/m³ fluid density - _s kg/m³ solids density - kg/m³ aggregate-suspension density difference - Ns/m² kinematic viscosity - amplitude of pulse ratio (ref. 23, 9) - s mean residence time - _s solids volume fraction     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

The mechanism of Na+ transport by rabbit urinary bladder

Summary The mechanism of Na⁺ transport in rabbit urinary bladder has been studied by microelectrode techniques. Of the three layers of epithelium, the apical layer contains virtually all the transepithelial resistance. There is radial cell-to-cell coupling within this layer, but there is no detectable transverse coupling between layers. Cell coupling is apparently interrupted by intracellular injection of depolarizing current. The cell interiors are electrically negative to the bathing solutions, but the apical membrane of the apical layer depolarizes with increasingI _sc. Voltage scanning detects no current sinks at the cell junctions or elsewhere. The voltage-divider ratio, , (ratio of resistance of apical cell membrane,R _a, to basolateral cell membrane,R _b) decreases from 30 to 0.5 with increasingI _sc, because of the transportrelated conductance pathway in the apical membrane. Changes in effective transepithelial capacitance withI _sc are predicted and possibly observed. The transepithelial resistance,R _t, has been resolved intoR _a, R_b, and the junctional resistance,R _j, by four different methods: cable analysis, resistance of uncoupled cells, measurements of pairs of (R _t, ) values in the same bladder at different transport rates, and the relation betweenR _t andI _sc and between andI _sc.R _j proves to be effectively infinite (nominally 300 k F) and independent ofI _sc, andR _a decreases from 154 to 4 k F with increasingI _sc. In the resulting model of Na⁺ transport in tight epithelia, the apical membrane contains an amiloride-inhibited and Ca⁺⁺-inhibited conductance pathway for Na⁺ entry; the basolateral membrane contains a Na⁺–K⁺-activated ATPase that extrudes Na⁺; intracellular (Na⁺) may exert negative feedback on apical membrane conductance; and aldosterone acts to stimulate Na⁺ entry at the apical membrane via the amiloride-sensitive pathway.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Morphological aspects of the hypothalamic-hypophyseal system

Summary Enzyme-histochemical methods were used to study the metabolic activity of specialized ependyma of the ventrolateral walls and floor of the third ventricle in young male and female rats during the critical period of sexual differentiation of the hypothalamus (one week after birth). Histochemical tests were conducted for glutamic dehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase, glycerophosphate dehydrogenase and NADH₂-dehydrogenase. Enzyme activity was judged by cytospectrophotometry. All the data were treated statistically.It was found that the specialized ependyma of the ventrolateral wall and floor of the third ventricle (median eminence) in rats differed in their enzyme behaviour in males and females during the critical period of sexual differentiation of the hypothalamus. At the level of the arcuate nucleus ( ²-tanycytes) and the medial part of the median eminence ( ²-tanycytes) the ependyma was characterized by similar indices of metabolic activity in males and females in the decisive terms of the critical period (days 3, 5, and 7). On day 5 metabolic activity of these cells was reduced both in the males and in the females. Prominent sexual differences in the intensity of the enzyme reactions studied were noted in the ependyma of the lateral parts of the median eminence ( ¹-tanycytes) in the critical period. On day 5 metabolic activity of ¹-tanycytes was reduced in males and increased in females. It is suggested that these differences are caused by the receptor nature of ¹-tanycytes and suggest their implication in the mechanisms of sexual differentiation of the hypothalamus.     

Identification of surface exposed amino acids of isolated and membrane-bound CF1 by protease accessibility

In order to compare surface-exposed amino acids in isolated and membrane-bound CF₁ the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF₁ the N-terminal region of the subunit was found to be highy sensitive to proteases; the accessible peptide bonds included E17-G18, R21-E22, E22-V23, and K24-V25. Additional protease-attacked bonds in subunit were S86-S87, xE125-S126. and R127-L128. In the subunit of isolated CF₁ the bonds L14-E15 and V76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF₁ computed after the crystal structure of mitochondrial F₁ by S. Engelbrecht (1997). In membrane bound CF₁ the primarily accessible peptide bond of the N-terminal domain of is R21-E22. After this bond is cleaved by trypsin, the K24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds R14O-S141 and G16O-R161 are cleaved. According to the Engelbrecht model G16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF₁. The subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF₁. In the subunit of membrane-bound CF₁ a papain-sensitive bond G102-G103 was identified. The results indicate major structural alterations when CF₁ is extracted from the CF₀CF₁ complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between C199 and C205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular G160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.     

Sequence divergence of B2m alleles of wild Mus musculus and Mus spretus implies positive selection

Mouse ₂-microglobulin (₂m) is polymorphic. Sequences of five allelic wild mouse B2m genes have been determined from the large exons of genomic DNA using the polymerase chain reaction. Relative to the standard B2m ^a allele, the products of four alleles of Mus musculus origin (w2, w3, w4, and w5), differ by only one or two amino acids. w5 has a single nucleotide change, Asp85 Val, and is identical to the c allele. w2 differs at Arg81 Thr and w4 at His34 Gln, and they share the Asp85 Val change with B2m ^c and B2m ^w5.w5 and c cells are lysed by S19.8, a monoclonal antibody specific for 2m^b (Ala85), in a complement-mediated cytotoxicity assay, whereas w4 cells are not. Thus, distant changes appear to introduce subtle conformational effects on 2m structure. Five independent isolates of Mus spretus (w1) differ the most from B2m ^a, with 12 amino acid changes and only one silent substitution. Replacements predicted from the nucleotide sequence occur in loops of the molecule facing away from the class I heavy chain and not in regions where 2m associates with class 1 3 domains. Concordantly, the w1 – 5 allelic forms of 2m associate well with H-2 heavy chains. The many amino acid changes in the spretus sequence and the paucity of silent substitutions suggest that B2m has been subject to positive selection.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M84362-M84367 and L04992-L04994.     

Photosynthetic ATPases: purification,properties, subunit isolation and function

Photosynthetic coupling factor ATPases (F₁-ATPases) generally censist of five subunits named , , , and in order of decreasing apparent molecular weight. The isolated enzyme has a molecular weight of between 390,000 to 400,000, with the five subunits probably occurring in a 3:3:1:1:1 ratio. Some photosynthetic F₁ ATPases are inactive as isolated and require treatment with protease, heat or detergent in order to elicit ATPase activity. This activity is sensitive to inhibition by free divalent cations and appears to be more specific for Ca²⁺ vs. Mg²⁺ as the metal ion substrate chelate. This preference for Ca²⁺ can be explained by the higher inhibition constant for inhibition of ATPase activity by free Ca²⁺. Methods for the assay of a Mg-dependent ATPase activity have recently been described. These depend on the presence of organic solvents or detergents in the reaction mixture for assay. The molecular mechanism behind the expression of either the Ca- or Mg-ATPase activities is unknown. F₁-ATPases function to couple proton efflux from thylakoid membranes or chromatophores to ATP synthesis. The isolated enzyme may thus also be assayed for the reconstitution of coupling activity to membranes depleted of coupling factor 1.The functions of the five subunits in the complex have been deduced from the results of chemical modification and reconstitution studies. The subunit is required for the functional binding of the F₁ to the F₀. The active site is probably contained in the (and ) subunit(s). The proposed functions for the and subunits are, however, still matters of controversy. Coupling factors from a wide variety of species including bacteria, algae, C₃ and C₄ plants, appear to be immunologically related. The subunits are the most strongly related, although the and subunits also show significant immunological cross-reactivity. DNA sequence analyses of the genes for the subunit of CF₁ have indicated that the primary sequence of this polypeptide is highly conserved. The genes for the polypeptides of CF₁ appear to be located in two cellular compartments. The , and subunits are coded for on chloroplast DNA, whereas the and subunits are probably nuclear encoded. Experiments involving protein synthesis by isolated chloroplasts or protein synthesis in the presence of inhibitors specific for one or the other set of ribosomes in the cell suggest the existence of pools of unassembled CF₁ subunits. These pools, if they do exist in vivo, probably make up no greater than 1% of the total CF₁ content of the cell.Abbreviations AMP-PNP adenylyl 5 imidodiphosphate - bchl bacteriochlorophyll - CF₁ chloroplast coupling factor 1 - CF₁-CF₀ the chloroplast ATP synthase complex - chl chlorophyll - CvF₁ F₁ from Chromatium vinosum - DCCD N, N-dicyclohexyl carbodiimide - EF₁ the coupling factor 1 isolated from membranes of Escherichia coli - F₀ the hydrophobic, integral membrane portion of the ATP synthase - F₁ coupling factor 1, the extrinsic membrane portion of the ATP synthase - FSBA 5-p-fluorosulfonylbenzoyladenosine - K_d dissociation constant - k_i inhibition constant - k_ii intercept inhibition constant - k_is slope inhibition constant - LS large subunit of ribulose bisphosphate carboxylase - MF₁ mitochondrial coupling factor 1 - M1F₁ F₁ from Mastigocladus laminosus - NBD-Cl 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - PAGE polyacrylamide gel electrophoresis - RcF₁ F₁ from Rhodopseudomonas capsulata - RpF₁ F₁ from Rhodopseudomonas palustris - RrF₁ F₁ from Rhodospirillum rubrum - RsF₁ F₁ from Rhodopseudomonas sphaeroides - SDS sodium dodecyl sulfate - S1F₁ F₁ from Synechococcus lividus - SpF₁ F₁ from Spirulina platensis - TF₁ F₁ from the thermophilic bacterium, PS3 - tricine N-tris (hydroxymethyl) methyl glycine - tris tris (hydroxymethyl)-amino methane; and - Vmax maximal velocity or maximal activity     

In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls

Saturable and reversible in vitro binding of [¹⁴C]riboflavin was found to occur on subcellular, sedimentable particles from maize coleoptiles and Cucurbita hypocotyls. The K_D was ca. 6 M, the pH optimum was near 6.0, and the number of binding sites amounted to 0.1–0.5 M on a fresh-weight basis. When the reducing agent dithionite was present, riboflavin binding increased-the K_D was 2.5 M, and the pH optimum above 8.0. The binding was specific: flavin mononucleotide (FMN) and flavin adenosine-dinucleotide (FAD) bound less tightly to these sites than riboflavin and another major soluble flavin, the previously described riboflavin-analog FX, occurring in grass coleoptiles. These flavin-binding sites were localized on vesicles derived from plasmalemma and endoplasmic reticulum by analyzing sucrose and metrizamide density gradients and marker enzymes.Abbreviations CCO cytochrome-c oxidase - CCR NADH-cytochrome-c oxidoreductase - ER endoplasmic reticulum - FAD flavin-adenosinedinucleotide - FMN flavin mononucleotide - MOPS N-morpholino-3-propansulfonic acid - NADH reduced -nicotinamide dinucleotide - nKP n thousand times g pellet - NPA l-naphthylphthalamic acid - PM plasma membrane, plasmalemma - RBF riboflavin - IAA indoleacetic acid - BA benzoic acid     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Photosynthetic CO2 Fixation in the Second Leaf of Wheat Seedlings Grown at Different Conditions of Nitrogen Nutrition

The rates of photosynthetic ₂ assimilation were determined in fully expanded second leaves of 21-day-old wheat (Triticum aestivum L.) seedlings grown on media supplied with nitrate or ammonia and on a nitrogen-free medium (NO₃ ^–- or NH₄ ⁺-treatments and N-deficit treatment, respectively). The maximal quantum efficiency of photosynthesis was independent on conditions of nitrogen nutrition. When leaves were exposed to 0.03% ₂ and high-intensity light, the lowest photosynthetic rate was noted for N-deficit treatment and the highest rate was characteristic of NH₄ ⁺ treatment. The elevation of the ₂ concentration in the gas phase to 0.1% stimulated photosynthesis at high-intensity light in all treatments. The rate of ₂ uptake by the leaf of N-deficient seedlings increased with ₂ concentration to a larger extent than in other treatments and approached the ₂ uptake rate characteristic of the NO₃ ^– treatment. In plants grown on a nitrogen-free medium, the leaf accumulated lesser amounts of reduced nitrogen and higher amounts of starch, but the content of chloroplast protein corresponded to that of NO₃ ^– treatment. In the leaf of NH₄ ⁺-treated seedlings, the rate of ₂ assimilation was higher than in the leaf of NO₃ ^– treated plants, regardless of the composition of the gas mixture. The ammonium-type nutrition, as compared to the nitrate-type nutrition, elevated the amount of reduced nitrogen in the leaf and promoted accumulation of chlorophyll and protein, the chloroplast protein in particular.     

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation

The interaction of heme-free (^o) and heme-containing (^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^–1 s^–1. This was 100-fold more rapid than the reported rate of combination of ^h and ^h proteins. This encounter-like rate of semi--hemoglobin (^oh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled ^o protein superimposed over native ^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the ₁₁ interface, was apparently responsible for the enhanced association properties of the ^o protein.     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.