Showing papers in "Journal of Bioenergetics and Biomembranes in 1984"

Evidence for direct roles of calcium in photosynthesis

Abstract: Calcium may function directly in several aspects of photosynthesis. It appears to modulate activity of the phosphatase enzymes in the carbon reduction cycle and also to regulate chloroplast NAD+ kinase activity through a calmodulin-like protein. Some evidence supports a calcium function in the water-splitting complex, and other evidence indicates a reaction center function in photosystem II. Calcium in reaction center II may be tightly bound in chloroplasts and weakly bound in blue-green algal thylakoids. Free calcium concentration in stroma is probably less than 10(-6) M, although the absolute concentration is not yet known. Intrathylakoid calcium content is likely very high. Stromal calcium may regulate several enzyme activities, while intrathylakoid calcium may promote photosystem II constitutively. Results to date demonstrate the need for more attention to cation composition in studies of both light and dark reactions of photosynthesis, and the need to identify free calcium levels in chloroplasts.

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site.

Abstract: In order to determine whether polymorphic forms of the Ca2+ + Mg2+-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca2+ + Mg2+-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca2+ + Mg2+-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca2+ + Mg2+-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca2+ + Mg2+-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca2+-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca2+. Its lack of cross-reactivity with proteins that do not bind Ca2+ suggests that it has specificity for antigenic determinants that make up the Ca2+-binding sites in several Ca2+-binding proteins including the Ca2+ + Mg2+-dependent ATPase.

Properties of a transplasma membrane electron transport system in HeLa cells.

Abstract: A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.

A transmembranous NADH-dehydrogenase in human erythrocyte membranes.

Abstract: Evidence is presented for a transmembranous NADH-dehydrogenase in human erythrocyte plasma membrane. We suggest that this enzyme is responsible for the ferricyanide reduction by intact cells. This NADH-dehydrogenase is distinctly different from the NADH-cytochromeb 5 reductase on the cytoplasmic side of the membrane. Pretreatment of erythrocytes with the nonpenetrating inhibitor diazobenzene sulfonate (DABS) results in a 35% loss of NADH-ferricyanide reductase activity in the isolated plasma membrane. Since NADH and ferricyanide are both impermeable, the transmembrane enzyme can only be assayed in open membrane sheets with both surfaces exposed, and not in closed vesicles. The transmembrane dehydrogenase has affinity constants of 90 µM for NADH and 125 µM for ferricyanide. It is inhibited byp-chloromercuribenzoate, bathophenanthroline sulfonate, and chlorpromazine.

A link between transport and plasma membrane redox system(s) in carrot cells

Abstract: Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K+ (86Rb+) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K+ (86Rb+) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K+ (86Rb+).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K+ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K+ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K+.

Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids

Abstract: Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20–30 single-turnover flashes and 8 min of dark before addition of ADP, Pi, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and Pi, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H+ (mg chl)−1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequested amine group array may provide localized association-dissociation sites for proton movement to the coupling complex.

Nonequilibration of membrane-associated protons with the internal aqueous space in dark-maintained chloroplast thylakoids

Abstract: Isolated spinach thylakoids retain a slowly equilibrating pool of protons in the dark which are predominantly bound to buffering groups, probably amines, with low pKa values. We have measured the effects of permeant buffers, salts, sucrose, and uncouplers on the retention of the proton pool. Acetic anhydride, which reacts with neutral primary amine groups, was used to determine the protonation state of the amine buffering groups. It was previously shown by Bakeret al. that the extent of inhibition of photosystem II water-oxidizing capacity by acetic anhydride and the increase in derivatization by the anhydride are proportional to, and dependent on, the deprotonated state of the amine buffering pool. Therefore, acetic anhydride inhibition of water oxidation activity may be used as a measure of the protonation state of the amine buffering pool. By this method it is inferred that protons, in a metastable state, were retained by membranes suspended in high pH buffer for several hours in the dark. When both the internal and external aqueous phases were equilibrated with pH 8.8 buffer, the proton pool was released only upon addition of a protonophore. The osmotic strength of the suspension buffer affected uncoupler-induced proton release while ionic strength had little influence. The acetic anhydride-sensitive buffering group(s) of the water-oxidizing apparatus had an apparent pKa of 7.8. We conclude that an array of protein buffering groups reside either within the membrane matrix, or in proteins at the membrane surface, not in equilibrium with the bulk aqueous phases, and is responsible for the retention of the proton pool in dark maintained chloroplasts.

Inhibition of plasma membrane NADH dehydrogenase by adriamycin and related anthracycline antibiotics.

Abstract: Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed. This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10−6–10−7 M adriamycin inhibit plasma membrane redox reactions >50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps

Abstract: The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O2 in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of "coupling relationships" between steps were designated as "sequential" and "fixed," respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochrome bc1 reactions, and between the cytochromes bc1 and aa3 reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between the bc1 and aa3 reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which the bc1 and aa3 reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of the bc1 and aa3 reactions observed previously by others. It is suggested that cytochrome c is the freely diffusible intermediate between the bc1 and aa3 reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochrome c and the cytochromes bc1 and aa3 complexes.

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum.

Abstract: Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na3VO3 as first described by Dux and Martonosi. These crystals exhibit repeat rows ∼11 nm apart which contain discrete units with ∼7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism.

Abstract: Two interesting previously reported properties of mitochondrial F1 ATPase have been confirmed and have been examined by 18O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO3- to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO3- was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 microM. The HCO3- activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each Pi formed when 5 or 10 microM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO3- acceleration of ADP departure after a binding change that stops 18O exchange and loosens ADP binding. When ITP concentration was lowered during net ITP hydrolysis by F1 ATPase an increase in water oxygen incorporation into Pi formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [gamma-18O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each Pi formed from the ATP. These results add to evidence for the binding change mechanism for F1 ATPase with sequential participation of catalytic sites.

Local anesthetics: a new class of partial inhibitors of mitochondrial ATPase.

Abstract: The following characteristics are reported for mitochondrial ATPase prepared by the chloroform extraction method: (1) The pH optimum for enzyme activity is at 8.0. (2) The neutral anesthetic benzocaine inhibits the enzyme at all pH values. (3) Reciprocal plots of 1/v versus 1/[ATP] show that inhibition by lidocaine, tetracaine, dibucaine, and chlorpromazine is noncompetitive; slope and intercept replots are hyperbolic, showing that the inhibition is partial rather than complete.

Interactions in cytochrome oxidase: functions and structure.

Abstract: Mitochondrial cytochromec oxidase is an exceedingly complex multistructural and multifunctional membranous enzyme. In this review, we will provide an overview of the many interactions of cytochrome oxidase, stressing developments not covered by the excellent monograph of Wikstrom, Krab, and Saraste (1981), and continuing into early 1983. First we describe its functions (both in the nominal sense, as a transporter of electrons between cytochromec and oxygen, and in its role in energy transduction). Then we describe its structure, emphasizing the protein (its structure as a whole, the number and stoichiometry of its subunits, their biosynthetic origin, and their interactions with each other, with other components of the enzyme complex, and with the membrane as a whole). Finally, we present a model in which the protein conformation serves as the focus for the dynamic interaction of its two major functions.

Phosphate transport, membrane potential, and movements of calcium in rat liver mitochondria.

Abstract: The membrane potential and calcium accumulation of mitochondria were followed by ion-specific electrodes in the presence of the proton-donor anions phosphate, acetate, glutamate, and beta-hydroxybutyrate. Phosphate was the only anion which allowed rapid and complete restoration of both the membrane potential and the steady-state extramitochondrial calcium concentration after the uptake of 100–200 nmol calcium per mg protein. If there was no influx of any proton-donor anion, the extent of calcium uptake depended on the intramitochondrial phosphate content. Both the fall of the membrane potential and the increase of the external calcium concentration brought about by a given amount of uncoupler were counteracted by phosphate transported into the mitochondria.

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: studies of its binding to F1 and its function.

Abstract: The binding of “oligomycin sensitivity conferring protein” (OSCP) to soluble beef-heart mitochondrial ATPase (F1) has been investigated. OSCP forms a stable complex with F1, and the F1 · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted submitochondrial particles. The F1 · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F1 is inactivated by 90% or more. Both free F1 and the F1 · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their β subunits; whether α subunits are also lost is uncertain. The cold-treated F1 · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F1- and OSCP-depleted particles. OSCP also protects F1 against modification of its α subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F1 cannot bind OSCP indicates that OSCP binds to the α subunit of F1 and that F1 contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F1 with the membrane sector of the mitochondrial ATPase system.

Effects of quinine on K+ transport in heart mitochondria.

Abstract: Quinine inhibits the respiration-dependent extrusion of K+ from Mg2+-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K+ and Na+ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na+ or K+ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent42K+/K+ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K+ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K+/H+ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.

On the mechanism of H+ translocation by mitochondrial H+ -ATPase. Studies with chemical modifier of tyrosine residues.

Abstract: In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+-ATPase complex. These effects are caused by nitration of tyrosine residues of H+-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles. H+ conduction in phospholipid vesicles inlaid with F0 is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F0 are critically involved in energy-linked proton translocation in the ATP-ase complex.

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase

Abstract: The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimirystoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18–28°C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots ofK m also show breaks in the same temperature interval, with the exception of the dioleoylenzyme vesicles, whereK m is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24°C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3–18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

Abstract: We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho degree) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized beta-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent.

Abstract: The thermodynamic efficiency of the Ca2+-Mg2+-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca2+ gradient established with the ATP/(ADP*Pi) ratio. The evaluation was made at an external Ca2+ level (4.7 × 10−8 M) which is below theK m value of 7 × 10−8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca2+ concentration of 17 mM was observed at infinite ATP/(ADP*Pi) ratio (absence of ADP). This corresponds to a [Ca2+]i/[Ca2+]0 gradient of 3.6 × 105. A Ca2+ gradient one-half as large was observed at an ATP/(ADP*Pi) ratio of 3.5 × 103 M−1. The square of the Ca2+ gradient is shown to be proportional to the ATP/(ADP*Pi) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 106 M) under these conditions (0.1 mM Mg2+, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.

The protein composition of the cytoplasmic membrane of aerobically and anaerobically grown Escherichia coli

Abstract: The protein composition of the cytoplasmic membranes ofEscherichia coli, grown aerobically and anaerobically on a glucose minimal medium at pH 70, were analyzed by crossed immunoelectrophoresis Qualitative differences are limited to only two proteins: nitrate reductase (EC 17994) is absent under aerobic growth conditions, whereas an unidentified protein, with a molecular weight of 81,500 and located at the inner side of the cytoplasmic membrane, is synthesized only in the presence of oxygen Quantitative differences are observed for many proteins: the ratio of the amount of a specific protein present in cells grown anaerobically and aerobically was, for four proteins, between 03 and 1; for 25 proteins, between 1 and 3; and for five proteins, larger than 5

Three proton pumps, morphology and movements.

Abstract: The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the ΔpH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 µg per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane.

Abstract: Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W · m−2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W · m−2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W · m−2, the ratio was approximately 35% lower than that of plants grown at 60 W · m−2. The amount of both cytochromeb563 and cytochromef was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.

Ionic requirements for taurocholate transport in rat liver plasma membrane vesicles.

Abstract: As part of the enterohepatic circulation, taurocholate is taken up by hepatocytes by a Na+-gradient-dependent, carrier-mediated process The dependence of taurocholate uptake on the presence of a Na+ gradient, outside greater than inside, has been studied in isolated rat liver plasma membranes The uptake is specific for sodium, and a cotransport stoichiometry of 2 Na+ per taurocholate taken up was found The presence of K+ ions inside the vesicles was also found to be essential for maximum Na+-stimulated uptake of taurocholate, although a K+ gradient is not required Mg2+ was almost as effective as K+ in this regard The symport of Na+ and taurocholate during uptake was shown to be electrogenic, so that K+ may act as an exchange counterion preventing the accumulation of positive charge within the vesicles

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase.

Abstract: Goat antisera against (Na+ + K+)-ATPase and its isolated subunits and against (K+ + H+)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na+ + K+)-ATPase antisera cross-reacted with (K+ + H+)-ATPase or inhibited its enzyme activity. The same was true for the (K+ + H+)-ATPase antiserum with regard to (Na+ + K+)-ATPase and its subunits and its enzyme activity. So notwithstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.

Activation studies by phospholipids on the purified cytochromec4:o oxidase ofAzotobacter vinelandii

Abstract: A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochromec4:o oxidase fromAzotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochromec4 (3.6 nmol/mg protein) and cytochromeo (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at “moderate” rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2–4-fold stimulation in TMPD oxidase activity occurred. Other “authentic” micellar phospholipids also readily activited TMPD oxidase activity in Fraction V. Themaximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.

Saturated amines and diamines as substrates which inhibit beef liver mitochondrial monoamine oxidase.

Abstract: Monoamines and diamines of 8–12 carbon atoms initially serve as substrates for purified beef liver monoamine oxidase but then lead to inhibition. The inhibition is not solely the result of aldehyde formation as addition of decylaldehyde does not inhibit benzylamine oxidation. Furthermore, neither the addition of alcohol dehydrogenase and NADH nor of semicarbazide prevent the inhibition of diaminodecane oxidation. The formation of a Schiff base on the enzyme surface resulting in aggregation or occlusion of the enzyme may be a cause of the inhibition. When concentrated enzyme solutions (≥1 mg/ml) are reduced by long-chain amines, 100% O2 causes only partial return of the flavin peak at 450 nm while enzyme activity continues to decrease. Substantial recovery of activity occurs (over a 3–4 week period) when inhibited enzyme is sedimented and resuspended in fresh buffer. These observations are discussed and compared with inhibition observed by other investigators with the substrate phenylethylamine.

Amino group modification of (Na++K+)-ATPase

Abstract: The effects of three amino group reagents on the activity of (Na++K+)-ATPase3 and its component K+-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na+ of Mg2+ present, the enzyme inactivation process follows complicated kinetics. In the presence of K+, Rb+, or Tl+, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg2+ (P 50 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na++K+)-ATPase. Mg2+ and K+ have no effects, and ATP only a minor effect, on the degree of modification. The K+-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na++K+)-ATPase activity. Half-inhibition of the (Na++K+)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na++K+)-ATPase. Mg2+ has no effect, and ATP has only a slight protecting effect. The K+-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na++K+)-ATPase activity. Half-inactivation of the (Na++K+)-ATPase activity is obtained after 20% modification of the amino groups.