The amino acid sequence of ferredoxin II from Chlorobium limicola, a photosynthetic green bacterium. The amino acid sequence of ferredoxin II from the photosynthetic green sulfur-reducing bacterium, Chlorobium limicola, was deduced to be: Ala-His-Arg-Ile-Thr-Glu-Glu-Cys-Thr-Tyr-Cys-Ala-Ala-Cys-Glu-Pro-Glu-Cys-Pro-Val-Asn-Ala-Ile-Ser-Ala-Gly-Asp-Glu-Ile-Tyr-Ile-Val-Asp-Glu-Ser-Val-Cys-Thr-Asp-Cys-Glu-Gly-Tyr-Tyr-Asp-Glu-Pro-Ala-Cys-Val-Ala-Val-Cys-Pro-Val-Asp-Cys-Ile-Ile-Lys-Val. The ferredoxin was shown to consist of 61 amino acids in a single polypeptide chain. The presence of 8 g-atoms of Fe and 8 mol of sulfide led to a calculated molecular weight of 7289. In constract to the ferredoxin I from C. limicola, ferredoxin II contains basic amino acids in positions 2 and 3 and 60 from the NH(2)-terminal end of the protein. The sequences of all the various ferredoxins from photosynthetic bacteria reported to date are compared with one another.


New method for isolation and sequence determination of 5'-terminal regions of bacteriophage phiX174 in vitro mRNAs. We have determined the nucleotide sequences of the 5'-terminal oligonucleotides, produced by RNase T1 digestion of bacteriophage phiX174 mRNAs synthesized in vitro. The major sequences are: pppCpGp(Ap), pppApUpCpGp(Cp), pppAp(Ap)2UpCp(Up)2Gp(Gp), and pppAp(Ap)3UpCp(Up)2Gp(Gp). The sequences of several minor 5'-terminal oligonucleotides were also determined. For this research we have devised a simple isolation procedure, for the 5'-terminal oligonucleotides, based upon hydroxylapatite chromatography and two-dimensional thin-layer separation. This method allows for the rapid and quantitative recovery of all oligonucleotides containing 5'-triphosphate end groups and should be generally useful for sequence on 5' termini of mRNAs.


Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.


Deuterium isotope effects during formation of phenols by hepatic monoxygenases. Evidence for an alternative to arene oxide pathway. The in vivo and in vitro metabolisms of normal and deuterated aromatic substrates have been investigated in ratsmsignificant isotope effects (k(H)/k(d) = 1.3-1.75) were associated with in vivo formation of meta-hydroxylated metabolites from 1:1 mixtures of normal and perdeuterio-(aryl ring) nitrobenzine, methyl phenyl sulfide, and methyl phenyl sulfone. Since isotope effects of this magnitude are incompatible with arene oxides as intermediates in the formation of phenols, the results provide evidence that multiple pathways are responsible for the formation of phenols in mammals. Significant isotope effects were not associated with the formation of the other phenolic isomers of nitrobenzene, methyl phenyl sulfone, or methyl phenyl sulfide or with the formation of phenolic products from anisole, bromobenzene, chlorobenzene, fluorobenzene, benzonitrile, naphthalene, zoxazolamine, acetanilide, biphenyl, diphenylhydantoin, benzene, o- and p-xylene, toluene, and mesitylene. Significant isotope effects might not be observable with the latter substrates if the kinetic parameters for oxidation of substrate change or if the arene oxide pathway greatly predominates. Furthermore, extensive in vivo metabolism of any substrate would make isotope effects unobservable by the procedure employed, namely the analysis of isotope content in metabolites formed from 1:1 mixtures of normal and deuterated substrates.


The nature of metal-ligand bonding in the primary hydration shell and in ionophores with alkali metal ions. 1. Trasport of alkali ions in model membranes mediated by ionophores is associated with at least two basic problems: (a) a conceivable physical model for the metal-ligand bonding in the primary hydration shell and (b) migration of ions from the primary hydration shell to ionophores to form ion complexes. 2. The nature of metal-ligand bonding both in the primary hydration shell and in the ionophores has been found to be around 80% ionic and 20% covalent in both cases by indepdnent investigation. Clearly the energetics being equivalent in both systems, ions will not migrate from the former to the latter. 3. A novel model is proposed in terms of hydrogen bonding in the primary hydration shell for Li+ and Na+ so that the migration of these ions to ionophores is easily envisaged. Hydrogen bonding contribures 18% and 5% of the energy to the total hydration shell energy of Li+ and Na+, respectively.


Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes. The exchange of phosphatidylcholine between (32P)phosphatidylcholine lipososomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of (23P)phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of the initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homofwnwoua pool od amLL single walled vesicles. Treatment of phosphatidyl (14C) choline liposomes with phospholipase D (phosphatidylcholine phosphantidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed cound not by hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of phosphatidylcholine from the inner to the outer layer of the liposome bilayer.


Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and Triton X-100. Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent. The early observation that the aminopeptidase form released from pig bursh border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papin. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.


The effect of pressure and the volume of activation on the monovalent cation and glucose permeabilities of liposomes of varying composition. Measurement of the cation permeability of phospholipid microvesicles as a function of pressure confirmed that a single rate-determining step occurred in each case. The volume of activation was 20 ml-mole minus 1 for Na+ and K+, and about 40 ml-mole minus 1 for valinomycin-mediated K+ permeability. It was virtually independent of membrane composition. The results were explained in terms of Träuble's theory of kink-substrate dissociation at the membrane interface involving possible 2gl and 2g2 kink isomers. The volume of activation for D-glucose was 37 ml-mole minus 1, which was not significantly different from that for any of the valinomycin-mediated K+ permeabilities. However, other data suggest that the rate-limiting steps for the sugar and cation permeabilities are not the same.


The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IX. The role of cellular calcium in the activation of the glucose transport system in rat soleus muscle. 1. The role of cellular Ca2+ in the transport of glucose has been investigated by determining the time-course of tension development and the release of 45Ca and 3-0-[14C]methylglucose from preloaded rat soleus muscles. 2. Electrical stimulation, 2,4-dinitrophenol (0.05 mM) and hyperosmolarity (200 mM mannitol) were all found to induce a rapid rise in tension and the rate coefficient of 45Ca release, which coincided with an acceleration of 3-0-[14C]methylglucose efflux. 3. Caffeine (10 mM) or exposure to K+ -substituted buffer induced a rapid increase in tension and the release of 45Ca, but a much later stimulation of 3-0-methylglucose efflux. This delayed response may be related to the fact that both factors induce a pronounced suppression of the effect of various agents known to stimulate sugar transport.4. Following a washout period of 120 min at 0 degreesC, the return to 30 degrees C elicited a prompt transient rise in the rate coefficient for the release of 45Ca and 3-0-[14C]meth ylglucose to levels, respectively, 2.8 and 14.6 times the control levels measured at 30 degrees C. The magnitude of these peaks appeared to be a function of the duration of the exposure to 0 degrees C. Cooling also led to a stimulation of the uptake of 3-0-[14C]methylglucose, and phlorizin suppressed the rise. 5. It was not possible to detect any significant effect of insulin on basal tension or on the influx or efflux of 45Ca. However, in a hyperosmolar environment, insulin (10-100 munits/ml) induced a marked further rise in tension, indicating that the hormone can elicit a redistribution of cellular Ca2+. 6. It is concluded that a rise in the cytoplasmic concentration of free Ca2+ constitutes a part of the mechanism by which the glucose transport system is activated by a variety of stimuli, perhaps also insulin.


An electrogenic component of the potential difference in the rabbit lens. The normal resting potential of the rabbit lens, -70mV, is altered to -59mV by ouabain concentrations up to 5-1- minus 6M, and to -52mV at 4 degrees C. Ouabain acts only at the anterior lens surface. The temperature effect id completely reversible. The Hodgkin-Katz-Goldman equation can be used with the measured lens potentials and Na+ and K+ levels in the lens and bathing medium to obtain alpha, the ration of the membrane permeabilities to Na+ and K+. The alpha-values obtained were 0.052 at 4 degrees C and 0.053 in 5-10 minus 6M ouabain. These data suggest that the change in potential due to cold and ouabain is caused by an inhibition of an electrogenic Na+ ump in the anterior lens epithelium.


Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. The quenching action of dibromothymoquinone on fluorescence and on primary photochemistry was examined in chloroplasts at minus 196 degrees C. Both the initial (F0) and final (FM) levels of fluorescence as well as the fluorescence of variable yield (FV equals FM minus FO) were quenched at minus 196 degrees C to a degree which depended on the concentration of dibromothymoquinone added prior to freezing. The initial rate of photoreduction of C-550 at minus 196 degrees C, which was assumed to be proportional to maximum yield for primary photochemistry, phipo, was also decreased in the presence of dibromothymoquinone. Simple theory predicts that the ratio FV/FM should equal phipo. Excellent agreement was found in a comparison of relative values of phipo with relative values of FV/FM at various degrees of quenching by dibromothymoquinone. These results are taken to indicate that FO and FV are the same type of fluorescence, both emanating from the bulk chlorophyll of Photosystem II. Dibromothymoquinone appears to create quenching centers in the bulk chlorophyll of Photosystem II which compete with the reaction centers for excitation energy. The rate constant for the quenching of excitation energy by dibromothymoquinone is directly proportional to the concentration of the quencher. Rate constants for the de-excitation of excited chlorophyll molecules by fluorescence, kF, by nonradiative decay processes, kD, by photochemistry, kP, and by the specific quenching of dibromothymoquinone, kQ, were calculated assuming the absolute yield of fluorescence at FO to be either 0.02 or 0.05.


Fluorescence quenching in photosystem II of chloroplasts. A simple photochemical model for the photosynthetic units of Photosystem II based on first-order rate constants for de-excitation of excited chlorophyll molecules is presented in the form of equations which predict the yields of fluorescence (i.e. at the FO level, at the maximal FM level and the fluorescence of variable yield, FV equals FM minus FO). Two types of quenching mechanisms are recognized: (1) increasing nonradiative decay processes in the bulk chlorophyll by creating quenching centers which complete with the reaction centers for the excitation energy (this mechanism quenches both FO and FV) and (2) increasing nonradiative decay of the excited reaction center chlorophyll (this mechanism quenches FV but not FO). Quenching in the bulk chlorophyll preserves the relationship that Fv/FM is equal to the maximum yield of photochemistry; quenching at the reaction center chlorophyll decreases FV/FM substantially (since FV is quenched specifically) but may have very little effect on the yield of photochemistry. Estimates are made of the relative magnitudes of the rate constants for de-excitation of the excited reaction center chlorophyll by photochemistry, kp, by nonradiative decay processes, kd, and by energy transfer back to the bulk chlorophyll, kt. Fluorescence is assumed to emanate only from the bulk chlorophyll. Energy transfer from Photosystem II to Photosystem I may occur from either the excited bulk chlorophyll or from the excited reaction center chlorophyll. The model is valid for any degree of energy transfer between Photosystem II units.


Effects of sodium and magnesium cations on the "dark-" and light-induced chlorophyll a fluorescence yields in sucrose-washed spinach chloroplasts. The effects of Na plus and Mg-2 plus on the "dark" level (O level) and light-induced (P level) fluorescence in sucrose-washed spinach clhoroplasts were studied. Low concentrations of NaCl (2-10 mM) cause a significant decrease in both the O and P levels in the chlorophyll fluorescence transient. The effect on the O level may reflect changes in the bulk chlorophyll a. At 77 degrees K NaCl increases the F735/F685 emission peak ratio in dark-adapted and preilluminated chloroplasts, but has no significant effect on this ratio in sucrose-washed Photosystem II particles. This evidence is consistent with a sodium-induced excitation-energy distribution in favor of Photosystem I. In the presence of MgCl2, with or without NaCl, there is a slight decrease in the O and P level fluorescence as compared with the salt-free control, but an increase as compared with the NaCl-treated sample. Magnesium appears to override the sodium-induced changes. At low temperatures in chloroplasts and Photosystem II particles, MgCl2 has different effects on the F735/F685 ratio apparently depending on the state of the membrane. Magnesium, however, always induces an increase in the F695/F685 ratio. These results suggest that magnesium may influence Photosystem II reaction centers as well as energy distribution between the two photosystems.


Temperature and preillumination dependence of delayed fluorescence of spinach chloroplasts. Delayed fluorescence (luminescence) from spinach chloroplasts, induced by short saturating flashes, was studied in the temperature region between 0 and minus 40 degrees C. At these temperatures, in contrast to what is observed at room temperature, luminescence at 40 ms after a flash was strongly dependent, with period four, on the number of preilluminating flashes (given at room temperature, before cooling). At minus 35 degrees C luminescence of chloroplasts preilluminated with two flashes (the optimal preillumination) was about 15 times larger than that of dark-adapted chloroplasts. The intensity of luminescence obtained with preilluminated chloroplasts increased steeply below minus 10 degrees C, presumably partly due to accumulation of reduced acceptor (Q minus), and reached a maximum at minus 35 degrees C. In the presence of 50 mM NH4Cl; at temperatures below minus 20 degrees C luminescence at 40 ms was decreased by NH4C1. At room temperature a strongly enhanced 40-ms luminescence was observed after the third and following flashes. The results indicate that both the S2 to S3 and the S3 to S4 conversion are affected by NlH4Cl. Inhibitors of Q minus reoxidation, like 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, did only slightly affect the preillumination dependence of luminescence at sub-zero temperatures if they were added after the preillumination. This indicates that these substances by themselves do not accelerate the deactivation of S2 and S3.


Salts and chloroplast fluorescence.

Salts and chloroplast fluorescence. Chlrorplast fluorescence was exicted by a weak measuring beam. A time-separated actinic light was used to modify the redox states of Q which in turn induced a change in the fluorescence yield. In salt-depleted chloroplasts, fluorescence saturated at a low actinic light intensity. CaCl2 increased the "variable" fluorescence as well as the rate of ferricyanide-Hill reaction. With Tris-washed chloroplasts, Photosystem II donor couple, phenylenediamine and ascorbate, did not increase the fluorescnece to a large extent without the presence of CaCl2. It is suggested that salt-depletion inactivates the Photosystem II reaction center of chloroplasts.


Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye. Changes in the fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K+ diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K+. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.


Mitochondrial oxygen affinity as a function of redox and phosphate potentials. 1. The conditions under which mitochondria might catalyse a net reversal of oxidative phosphorylation are analysed. 2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen. 3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration. 4. The K-m for oxygen is less than 0.1 muM at low phosphate potential, irrespective of substrate, and 1-3 muM under reversal conditions. 5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action.


Cytochrome b and photosynthetic sulfur bacteria. Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome alpha-band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll). The b-type cytochrome in Chromatium has an alpha-band maximum at 560 nm and a midpoint oxidation-reduction potential of -5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an alpha-band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near -90 mV. Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na-2S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.


The magnetic susceptibility of reduced cytochrome P-450-cam. The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of Photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.


Enrichment of photosystem I reaction center chlorophyll from spinach chloroplasts. The reaction center chlorophyll of Photosystem I in spinach chloroplasts was highly enriched. Preparations having 5-9 chlorophylls per 1 P700 were obtained by treating the Photosystem I particles prepared by digitonin treatment of chloroplasts with wet diethyl ether. All P700 present in the extracted particles was found to be photoactive, undergoind oxidation upon illumination.


Mode of action of antitumour antibiotics. spectrophotometric studies on the interaction of chromomycin A3 with DNA and chromatin of normal and neoplastic tissue. The binding of chromomycin A3, an antitumour antibiotic, to various DNA and chromatin isolated from mouse and rat liver, mouse fibrosarcoma and Yoshida ascites sarcoma cells was studied spectrophotometrically at 29 degrees C in 10-2 M Tris-HCl buffer, pH 8.0, containing small amounts of MgCl2 (4.5-10-5--25-10-5 M). An isobestic point at 415 nm was observed when chromomycin A3 was gradually titrated with DNA/chromatin and its spectrum shifted towards higher wavelength. The rates and extent of these spectral changes were found to be dependent on the concentration of Mg2+. The change in absorbance at 440 nm was used to calculate apparent binding constant (Kap M-1) and sites per nucleotide (n) from Scatchard plots for various DNA and chromatins. As expected, values of n for chromatin (0.06-0.10) were found to be lower than found for corresponding DNA (0.10-0.15). Apparently no such correlation exists between binding constants (Kap M-1)-10-4) of DNA (6.4--11.2) and of chromatin (3.1--8.3), but Kap M-1 of chromatin isolated from mouse fibrosarcoma and Yoshida ascites sarcoma are 1.5--3 times higher than that found for mouse and rat liver chromatin. These differences may be taken to indicate structural difference in nucleoprotein complexes caused by neoplasia. The relevance of this finding to tumour suppressive action of chromomycin A3 is discussed.


The inhibition of nuclear RNA synthesis by the rifampicin derivative AF/013 in living cells. The rifampicin derivative, AF/013, completely inhibits synthesis of the nucleolar and chromosomal RNA in explanted salivary gland cells of Chironomus tentans. When the glands are preincubated in rifampicin AF/013 for a short period before the addition of the radioactive precursors, labelling of RNA is depressed in all size classes to the same extent. In contrast, if rifampicin is replaced by the nucleoside analogue, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a tentative initiation inhibitor of heterogeneous nuclear RNA, the label is reduced preferentially in the lower molecular weight region of the heterogeneous nuclear RNA spectrum. In chase type experiments, when rifampicin AF/013 is added after an initial labelling period, the synthesis of heterogeneous nuclear RNA is suppressed equally in all size classes, a result analogous to that obtained with the elongation inhibitor, alpha-amanitin. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, under similar chase conditions, preferentially inhibits the labelling of smaller heterogeneous nuclear RNA molecules, but later on abolishes labelling of molecules with higher S values, also. Rifampicin AF/013 prevents or affects seriously the normal processing of the prelabelled preribosomal RNA in the nucleolus. It further interferes with the export of nuclear RNA to the cytoplasm, and/or promotes a non-physiological breakdown of cytoplasmic RNA. The experimental data suggest that rifampicin AF/013 acts on RNA synthesis in living cells by interference with chain elongation.


Muscle protein biosynthesis in the tumour-bearing rat.  A defect in a post-initiation stage of translation. 1. The decreased ability of polysomes isolated from the gastrocnemius of rats bearing the Walker 256 carcinoma to incorporate L-[14C]leucine into protein persisted in the presence of 5-10-5 M aurin tricarboxylic acid and 1-10-2 M NaF. 2. Poly(U)-directed phenylalanine incorporation by such polysome preparations was less than that of similar preparations from normal rats. 3. The ability of gastrocnemius polysomes from tumour-bearing rats to react with puromycin was markedly decreased. Cycloheximide inhibited peptidyl puromycin formation by polysome preparations from both normal and tumour-bearing rats. 4. The ability of recombined 60 S and 40 S subunits prepared from polysomes of tumour-bearing rats to carry out poly(U)-directed polyphenylalanine synthesis was much reduced when assayed over a wide range of magnesium concentrations. Cross-over experiments with subunits from normal and tumour-bearing animals suggested that this was due to a defect in the smaller ribosomal subunit.


The control of protein synthesis during the stimulation of lymphocytes by phytohaemagglutinin. III. Poly(U) translation and the rate of polypeptide chain elongation. 1. Cell-free systems from phytohaemagglutinin-stimulated pig lymphocytes are much more active in both endogenous protein synthesis and the translation of poly(U) than those from unstimulated lymphocytes. 2. Addition of tRNA stimulates the translation of poly(U) and greatly reduces the difference between systems from stimulated and unstimulated lymphocytes. Endogenous protein synthesis is not increased by added tRNA. 3. Systems from stimulated lymphocytes have an increased capacity to form aminoacyl-tRNA with several different amino acids. This reaction is limited by the amount of tRNA present in the cell-free system. 4. The rate of polypeptide elongation is not affected by lymphocyte stimulation. The increased rate of protein synthesis must therefore be due to an increase in the frequency of initiation of the synthesis of protein molecules.


Synthesis of alpha- and beta-globin directed by messenger ribonucleoprotein from rabbit reticulocytes. The translation of globin messenger ribonucleoprotein (mRNP) obtained from high salt-washed rabbit reticulocyte ribosomes by treatment with EDTA was investigated using a cell-free system from mouse Krebs II ascites tumour cells. The messenger activity of the mRNP and the mRNA derived from it by mild deproteinization was compared in the presence and absence of reticulocyte initiation factors. Both forms gave identical products over a wide range of messenger concentration and there was no qualitative or quantitative difference in their efficiency as messengers. It is concluded that the proteins associated with polysomal mRNA do not alter the specificity of translation of alpha- and beta-globin messengers or the requirement for initiation factor.


Inhibition of macromolecular synthesis in L1210 mouse leukemia ascites cells by hycanthone. Hycanthone inhibits macromolecular synthesis in L1210 cells. At the same concentrations of hycanthone, DNA synthesis is inhibited to a greater extent than the synthesis of RNA. There is an inhibition by the drug of the synthesis of ribosomal precursor RNA; the synthesis of other types of RNA including heterogeneous and small-molecular-weight, also appears to be inhibited. Maturation of 45-S RNA does not seem to be affected, and there is a partial reversal of inhibition of both DNA and RNA synthesis by washing the cells free of hycanthone.


Deoxyribonucleic acid-dependent ribonucleic acid polymerases from spleen of uninfected and Rauscher murine leukemia virus-infected NIH Swiss mice.? The RNA polymerase activities from the nuclei of the spleen of uninfected and Rauscher murine leukemia virus-infected NIH Swiss mice were resolved by DEAE-cellulose column chromatography, and their properties were compared. The RNA polymerase activities from infected and uninfected spleens were the same with respect to column elution profiles, optimum requirements for various salts, ratios of activities with Mn2+ and Mg2+, sedimentation values, and response to most templates. With the exception of minor differences in activities with certain DNA templates, the significance of which is not clear, no qualitative differences in the enzymes from these two sources were found, but an increase in the specific activity of the alpha-amanitin sensitive enzyme, RNA polymerase II, was found in the leukemic spleen. These preliminary results suggest that there may be no novel RNA polymerase induced by Rauscher murine luekemia virus-infection, and they are in keeping with the interpretation that the viral DNA genome is transcribed by a host RNA polymerase.


DNA-dependent DNA polymerase activities from Paramecia macronuclei. Three DNA-dependent DNA polymerase activities, A, B, and C, have been detected from the macronucleus of Paramecium. The enzymes were separated and partially purified by DEAE-cellulose and phosphocellulose chromatography. These three enzymes can be further distinguished from each other by the effect of KCl, araCTP and different DNA templates on their activities. All three enzymes have the same molecular weight (900 000-110 000), are dependent on Mg2+ and added DNA template and are inhibited by sodium pyrophosphate. The relative amounts of the three enzyme activities vary with the growth stage of the Paramecia from which they are isolated. In balanced growth, three polymerase activities (A, B AND C) are observed, whereas in late log or stationary phase polymerase C is absent, while the level of polymerase B decreases by a larger factor than polymerase A. These results have been related to the possible functional roles of the three activities and their relationship to activities in other organisms studies is discussed.


Circular dichroism and fluorescence studies on potato virus X and its structural components. Circular dichroism (DC) measurements of the coat protein subunits of potato virus X show that native subunits that can reassemble with RNA to form infectious virus particles have appreciable alpha-helical structure. The CD of intact potato virus X was less intense below and more intense above 250 nm, and the maxima and minima were at longer wavelengths, than those of a CD spectrum computed from the individual contributions of the coat protein and RNA. The differences between the measured and computed spectra below 250 nm were attributed to the effects of differential light scattering and absorption flattening on measurements of the virus particle CD. The differences at longer wavelengths, were the CD contribution of the nucleic acid predominates, probably reflect the difference between a base-paired conformation of the RNA in solution and the more rigid single-stranded conformation imposed by the structure of the virus. The CD evidence suggests that the tertiary structure and potato virus X coat protein subunits in solution and in intact virus particles is similar. Both CD and fluorescence emission results indicate differences between the tryptophan environment in dissociated protein subunits and that in intact virus. These are attributed to local differences in subunit conformation or to the occurrence of intersubunit interactions involving tryptophan in the intact virus.


Isolation of five active thyrotropin components from human pituitary gland. A procedure is described for the isolation of human pituitary thyroid-stimulating hormone (thyrotropin). The starting material was a side-fraction provided by the earlier developed process for the purification of growth hormone from whole frozen pituitaries. This fraction was further purified by successive chromatography on Bio-Gel P-150, Bio-Gel HT hydroxyapatite, and SP-Sephadex C-50. The resulting preparation was obtained in yields of 10 mg/kg of pituitary tissue and had a thyrotropin potency of 11 units human Research Standard A/mg as measured by a specific radioimmunoassay. Contamination by other pituitary hormone activities was low. In the ultracentrifuge a single sedimenting boundary was registered with an s20,w value of 2.7 S. The molecular weight as determined by sedimentation-equilibrium experiments was 34 000 in phosphate buffer, pH 7.0, and 17 700 in 1 M propionic acid. This thyrotropin preparation was, however, electrophoretically heterogenous. Following preparative polyacrylamide gel electrophoresis five different components associated with thyrotropin activity were isolated. Isolation on a preparative scale of electrophoretically homogeneous human thyrotropin has not earlier been reported. One of the thyrotropin components was characterized with respect to molecular weight and amino acid composition. The data were consistent with a molecular weight of 33 000 from sedimentation-equilibrium analysis at pH 7 and with 268 amino acid residues per molecule.


Electron diffraction study of hydrated phospholipid single bilayers. Effects of temperature hydration and surface pressure of the "precursor" monolayer. The molecular packing and phase transition of hydrated dipalmitoylphosphatidylcholine single bilayers are studied by electron diffraction, using an electron microscope equipped with a hydration stage. The phase transition and area per molecule are measured as functions of temperature, hydration and the surface pressure of the monolayer from which the bilayer is formed. The transition temperature of a bilayer agrees with calorimetric measurements on bulk lipid/water mixtures. The molecular packing of a bilayer corresponds to that of the precursor monolyer at a surface pressure of 47 dyne/cm.


Light scattering spectroscopy of the squid axon membrane. Light scattering studies on the giant squid axon were done using the technique of optical mixing spectroscopy. This experimental approach is based on the use of laser light to detect the fluctuations of membrane macromolecules which are associated with conductance fluctuations. The light scattering spectra were similar to the Lorentzian-like behavior of conductance fluctuations, possibly reflecting an underlying conformational change in the specific membrane sites responsible for the potassium ion conductance. The amplitude of the spectra measured, increased when the membrane was depolarized and decreased on hyperpolarization. The spectra were fit to the sum of two terms, a (1/f component and a simple Lorentzian term. Spectra from deteriorating axons did not show sensitivity to membrane potential changes. It is shown theoretically that fluctuations due to the voltage-dependent variable, n, of the Hodgkin-Huxley formalism are identical to the voltage fluctuations. The derived power spectrum is that of a second order system, capable of showing resonance peaking only if the voltage dependence of the potassium rate of constants is included in the analysis. The lack of resonance peaking in the observed light scattering spectra, indicates that the data are best described by a damped second order system.


Concanavalin A-reactive protein of rabbit thymocyte plasma membranes: analysis by crossed immune electrophoresis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. 1. Thymocyte plasma membrane extracts, prepared with the non-ionic detergent Triton X-100, show 10 major protein components upon sodium dodecysulfate/polyacrylamide gel electrophoresis and at least 11 immunologic components upon crossed immune electrophoresis. 2. Concanavalin A reactive membrane proteins have been identified using crossed immune electrophoresis with receptor-ligand interaction. 3. These proteins are absorbed from Triton X-100-solubilized membranes onto immobilized concanavalin A. They are eluted in stepwise fashion, using increasing concentrations of alpha-methyl-d-glucoside, between 0.0004 M and 0.1 M. The predominant proteins eluted in each step are components with high electrophoretic mobility in crossed immune electrophoresis and are identical with a glycosylated component in sodium dodecysulfate/polyacrylamide gel electrophoresis with molecular weight of 55 000. 4. This component forms multimers in the presence of Triton X-100 which are not totally dissociated in sodium dodecylsulfate. 5. Neuramidase treatment followed by crossed immune electrophoresis of total plasma membrane isolates, as well as the purified glycoprotein fraction, indicates that the concanavalin A-reactive proteins are sialoglycoproteins. 6. Sodium dodecylsulfate component 5.1 comprises at least two different populations of glycoproteins (6 and 9) in crossed immune electrophoresis, one of which exclusively exhibits heterogenous carbohydrate antigenic sites (component 9). 7. Present data, taken together with previously published experiments, indicate that concanavalin A binding to intact thymocytes induces an increased turnover and release of the receptor protein(s).


Zero-trans and infinite-cis uptake of galactose in human erythrocytes. 1. The zero-trans and infinite-cis uptake of galactose into human erythrocytes was measured as a function of galactose concentration at 20 degrees C. 2. A special procedure, the "cis-trans test" has been developed to determine the directionality of an asymmetric transport carrier. 3. Using the "cis-trans test" and results obtained by phloretin inhibition, could show the existance of two sites mediating galactose uptake. The kinetic parameters of the high affinity site are K1 equals 11 mM; V1 equals 16 mmol - cell unit-1-min-1 and of the low affinity site: K2 equals 286 mM; V2 equals 21 mmol-cell unit-2-min-1. 4. The infinite-cis Km, using an intergrated rate equation treatment was 21 mM and that found by a direct preloading procedure was 25 mM. The existence of a high affnity site at the inner side of the membrane was thus confirmed.


Zero-trans and equilibrium-exchange efflux and infinite-trans uptake of galactose by human erythrocytes. 1. The zero-trans and equilibrium exchange efflux and the infinite-trans uptake of galactose in human erythrocytes were measured as a function of galactose concentration at 20 %. 2. The zero-trans procedure with cells loaded with 285 mM galactose revealed a low affinity site for galactose transport at the inner face of the membrane having a maximal velocity of 255 plus or minus 96 mmol/l isotonic cell water and Km equals 240 plus or minus 57, the V/K ratio being 1.01 plus or minus 0.04 min-1.  3. The equibirum-exchange procedure yielded a maximal velocity of 432 plus or minus 44 mmol/cell unit per min and K equals 138 plus or minus 57, the  V/K ratio being3.19 plus or minus 0.52 min-1. 4. The infinite-trans uptake revealed a high affinity site at the outer face of the membrane having a maximal velocity of 239 plus or minus 11 mmol/cell unit per min, and K equals 21 plus or minus 2 mM. 5. These results combined with previous findings (Ginsburg, H. and Stein, W. D. (1975) Biochim. Biophys. Acta 000, 000-000) force us to reject the following models for sugar transport in human erythrocytes: a single asymmetric carrier; two symmetric carriers in parallel, the original form of the internal transfer model.


Kinetics of blood-brain transport of hexoses. 1. The kinetics of transport of glucose and four other hexoses through the blood-brain barrier were studied with a tritiated-water reference technique in the anesthetized rat. Brain clearance of [14-C]hexose was measured 15 s after a single injection of the hexose and 3-HOH reference into the common carotid artery. 2. Saturation of brain clearance of [14-C]hexose conformed to Michaelis-Menten kinetics. Linear transformation of the uptake data yielded the Km of carrier-mediated hexose transport: 2-deoxy-D-glucose 6 mM, D-glucose 9mM, 3-O-methyl-D-glucose and D-galactose 40 mM. A maximum transport velocity of 1.56 mumol/g per min was calculated and shown to be constant for all five hexoses. 3. The kinetics of 3-HOH and 3-0-methyl-D-[14-C]glucose efflux from brain to blood were studied with a modification of the water reference technique. An estimate of cerebral blood flow, 0.56 ml/g per min, was made from the efflux rate constant for 3-HOH, 0.61 min-1. The fractional extraction of 3-0-methyl-D-[14C]glucose uptake from blood was estimated from the efflux rate constant, 0.22 min-1, for this sugar and found to be 0.25. This value approximated the fractional extraction of 3-0-methyl-D-[14-C]glucose uptake that was determined from influx studies (0.24). These results indicated that the bidirectional movement of glucose across the blood brain barrier was symmetrical, which suggested that barrier sugar transport is equilibrative and not active. 4. Blood-brain barrier sugar transport was shown to be reversibly inhibited by phloretin, yet no modulation of transport was demonstrable after 2 or 8 days of starvation. Finally, regional analysis (olfactory bulb, caudate-putamen nucleus, thalamus-hypothalamus, and inferior-superior colliculi) demonstrated that, in addition to blood-brain barrier permeability, brain clearance of glucose was a function of cerebral blood flow.


Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid? Cycloleucine accumulation by frog muscle was studied at 0 degrees C and 25 degrees C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 degrees C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 degrees C than at 25 degrees C. The steady state data are consistent with an adsorption model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model. Diffusion-limited flux is ruled out on the basis of "influx profile analysis" of the time course of cycloleucine entry at both 0 degrees C and 25 degrees C. At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 degrees C and 25 degrees C. The rate constant of efflux does not vary with cellular concentration. These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflux only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 degrees C and 25 degrees C. Therefore, an adsorption model for cycloleucine accumulation in frog muscle appears to be untenable.


Dielectric breakdown in the membranes of Valonia utricularis. The role of energy dissipation. The electrical properties of the membranes of Valonia utricularis were investigated using intracellular electrodes. Using short (0.5-1.0 ms) current pulses it was found that at a critical membrane potential difference of 0.85 V there was a large and discontinuous decrease in the membrane impedance and the slope resistance beyond this potential was virtually zero. The electrical breakdown of the membranes did not lead to global damage of the cells and after a resealing time of approx. 5 s could be repeated with identical results. Experiments with long current pulses and long bursts of pulses repeated at 1 kHz are described which show that the electrical breakdown is not due to thermal damage arising from localized heating in the membrane. Thus a dissipation of some 10-3-10-5 times the energy normally dissipated during the onset of breakdown did not lead to breakdown itself unless the critical membrane potential was exceeded. The results also show that punch-through and avalanche ionization are not likely to be important in the breakdown mechanism. The results are consistent, however, with there being a critical instability in the electro-mechanical stresses set up in the membrane at large electric field strengths.


On the molecular nature of chloroplast thylakoid membranes. Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were disintegrated and the electrophoretic behavior of the components studied with special regard to the pigment-protein complexes. The process of denaturation of the complexes was found to differ with respect to the other protein components. As the result of denaturation, the pigment-free protein moieties exhibit altered electrophoretic mobilities in relation to the intact complexes mainly conditioned by two processes contrary in their action, i.e. increase of change and change of the hydrodynamic properties. Exhaustive extraction of the thylakoid membranes with 6 M guanidine - HCl removes the proteins mainly associated by polar and weak hydrophobic interactions. The insoluble residue quantitatively exhibits the pigment-protein complexes including their denatured protein moieties, two extrinsic hydrophobic proteins as well as some protein traces. Electron-microscopic studies demonstrate the material still to have a high degree of order and preserved basic structure. After removing the lipids from the basic membrane, large amounts of the protein moeity of Complex II become soluble in guanidine -HCl. Since all other lamellar proteins are removable either by quanidine -HCl extraction or by trypsin digestion it is assumed the basic membrane of thylakoid to consist only of the pigment-protein complexes embedded into the lipid matrix.


(Ca-2+ + Mg-2+)-Activated ATPase in the plasma membrane mouse liver cells. 1. Purified plasma membranes from dissociated adult mouse liver cells posses a (Ca-2+ + Mg-2+)-stimulated ATPase (EC 3.6.1.3) activity. 2. Enzyme activity is at a maximum with the addition of 0.3 mM Ca-2+ and 3 mM Mg-2+. 3. Using medium devoid of alkali metal ions (Ca-2+ + Mg-2+)-ATPase enzyme activity was observed with Km1 = 0.35 - 10-3 M at a substrate concentration of 1 mM or less and an apparent Km2 = 0.88 - 10-3 M at higher substrate concentrations. 4. In the presence of Na+ and 4 mM ATP, an increase in activity was seen, suggesting the presence of a (Ca-2+ + Mg-2+ + Na+)-activated ATPase. 5. In the presence of both Na+ and K+ the (Ca-2+ + Mg-2+)-dependent enzyme activity was further increased, indicating that a (Ca-2+ +Mg-2+ + K+)-stimulated ATPase may also be present.


The proteins of the content of the secretory granules of the rat parotid gland. The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [3-H]proline-labeled proteins on DEAE-cellulose and by amino acid analysis. Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the alpha-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins. The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%). The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-se granules. This protein, however, differed from the "membranous" proline-rich proteins by several criteria. Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%). Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining. The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.


An analysis of the D-glucose influx kinetics of in vitro hamster jejunum, based on considerations of the mass-transfer coefficient. 1. A study designed specifically to investigate the effects of unstirred layers on the apparent glucose-influx kinetics of hamster jejunum was conducted. 2. The apparent V was 12.81, 10.71, 9.75, 10.17 and 9.33 mumol/cm-2 - h while the apparent Km was 7.42, 3.95, 1.87, 0.93 and 0.5 mM, respectively, when the rate of shaking the incubation flasks was 40, 80, 120, 160 and 200 cycles/min. 3. Extrapolation of the slope and reciprocal intercept of Lineweaver-Burke plots of the data to infinite shaking rate is mathematically justified to yield the slope and intercept of a Lineweaver-Burk plot which is uncomplicated by unstirred layers. These extrapolations were found to have a regression coefficient = 1 when plotted as (intercept)-1 or slope = b0 + b1b-(shake)-2 where b = 2.764 for the slope plot and 6.626 for the (intercept)-1 plot. From the values of b0 one obtains a Km of 0.41 and a V 0f 0.35 which should represent the true kinetic parameters for glucose influx into this tissue under the experimental conditions employed. 4. Values of the theoretical flux expected on a basis of unstirred-layer thickness which was calculated from the relation Cb (for J = V/2) = Km + 0.5 V/Kd agreed with the experimental values of J in some instances but the 95% confidence interval of the theoretical and experimental values did not overlap in many instances at low shaking rates and low concentrations of glucose. 5. A factor theta representing the error between the theoretical and experimental values was found to fit the relationship 1n(theoretical J) = - 3.8 + 5.77 (1/theta) with a regression coefficient of 0.98 and was proposed to be due to one or more of the following parameters: (1) a villus tip to base gradient of transport (influx) activity; (2) a dependence of brush-border influx area on substrate concentration in the bulk incubation media; and (3) an end-product inhibition of the overall transport rate. 6. It is apparent from the data that the flux of glucose across the unstirred layer is ordinarily the rate-limiting step in the trans-brush-border transport of this sugar by hamster jejunum when less than saturating concentrations of glucose are used. At high shaking rates the contribution of the unstirred layer is reduced.


A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes. Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface proteins by 4.4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glucoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two proteins of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substantial differences in reactivity of proteins of the outer surface of the membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.


Effects of Tris and histidine on human erythrocytes and conditions influencing their mode of action. 1. The incubation of human erythrocytes in 0.172 M Tris - HCl, pH 7.6 buffer at 37 degrees leads to (1) a pronounced cellular volume increase, (2) a preferential release of Na+, and (3) if continued sufficiently long, hemolysis. These effects are pH dependent and also are influenced to a considerable degree by such diverse reagents as NaC glucose, and histidine. In each instance, increasing levels of the latter compounds in a Tris - HCl incubation mixture led to diminished cellular volume increase and prolonged time of onset of hemolysis. 2. Histidine solutions of 0.31 M, pH 7.5 caused a rapid and dramatic decrease in cellular volume of human erythrocytes and a concomitant rapid exit of cations. However, in a prolonged incubation, human erythrocytes slowly regained their cell volume as a result of histidine entry into the cell. Of considerable interest: Tris swollen cells undergo immediate shrinkage to far below the initial cell volume when incubated in histidine at 37 degrees C. Through repetition of this process two additional times, as much as 90-95% of the total cellular Na+ and K+ was removed without hemolysis. 3. Human erythrocytes washed in 0.12 M MgCl2 and then suspended in 0.31 M histidine, pH  7.5, lost upwards of 60% of their total Na+ and 30% of their total K+ after a 40 min incubation at 37 degrees C. However, when increasing amounts of 0.172 M Tris - HCl, pH 7.6 were added to the histidine suspension of cells, the release of K+ was reduced to 5% but the release of Na+ decreased only to 40% of the total cellular level. On the basis of these observations, it is evident that Tris exerts a preferential activity towards the efflux of Na+ from the human erythrocyte, whereas histidine results in high efflux of K+ and Na+ from the cell.


Heterogeneous elevation of amino acid transport rates in pantothenate-and lipid-deficient Lactobacillus plantarum. The effect of a pantothenic acid deficiency in Lactobacillus plantarum on the initial rate of amino acid transport was investigated. Although the steady-state accumulation capacity for all amino acids was markedly reduced in pantothenate-deficient cells, initial rates of uptake either were not changed (asparagine, alanine, lysine) or were increased (glutamic acid, aspartic acid leucine). The findings suggest that a reduction in membrane lipid content heterogeneously affects the operation and/or synthesis of amino acid transport catalysts.


Application of pulse radiolysis to the study of drug interactions with biological macromolecules. Pulse radiolysis has been used to study the interactions of a variety of small molecules with DNA. The diffusion-controlled rate constants for the reactions of e(aq) with dyes, cations and other small molecules including radioprotectors and radiosensitizers, were found to decrease remarkably upon association with DNA. These kinetic data can be used to quantitate the degree and extent of such interactions. This technique may have wide practical application for the in situ study of many small molecule-macromolecule associations, under physiological conditions.


Biochemical aspects of the visual process.  XXVII. Stereospecificity of ocular retinol dehydrogenases and the visual cycle. A comparative study is made of the stereospecificity of two particulate retinol dehydrogenases from bovine eyes and of horse liver alcohol dehydrogenase.  The particulate retinol dehydrogenase of outer segments reacts with the all-trans isomers of retinaldehyde and retinol but not with the 11-cis  compounds.  In contrast, a particulate retinol dehydrogenase present in pigment epithelium reacts preferentially with the 11-cis compounds.  Horse liver alcohol dehydrogenase (EC 1.1.1.1.) can convert both isomers, but the all-trans isomers are clearly preferred.  Differences with regard to cofactor preference and stability are also noted.  The outer segment enzyme clearly functions in the rhodopsin cycle.  It is unlikely that the 11-cis retinol dehydrogenase from pigment epithelium is directly involved in providing 11-cis retinaldehyde from rhodopsin regeneration, but it may serve to make available 11-cis retinaldehyde from rhodopdsin, digested in phagocytized rod sacs, for the synthesis of visual pigment by the visual cells.


De novo synthesis and developmental control of the multiple gene-controlled malate dehydrogenase isozymes in maize scutella. In maize, both soluble and mitochondrial malate dehydrogenases (L-malate:NAD oxidoreductase, EC 1.1.1.37)exist in multiple molecular forms (isozymes). Biochemical studies and genetic analysis showed that the maize malate dehydrogenase isozymes are not interconvertable conformational forms of a single gene product. Instead, they are determined by multiple genes which may reside on different chromosomes. Therefore, developmental expression of the various malate dehydrogenase isoqymes was studied. During early development of the sporophyte (dry kernel to 10 days of germination), the total malate dehydrogenase activity in scutella increases through the first 5 days, peaks about the 6th day and decreases gradually thereafter. Both soluble and mitochondrial malate dehydrogenase isozymes exhibit similar activity profiles; however, the total mitochondrial malate dehydro genase activity is only 60% of that in the cytosol. Density labeling experiments and atempts to detect the possible existence of "inactive malate dehydrogenase precursors" were performed. The results show that both soluble and mitochondrial malate dehydrogenase are synthesized in the scutella during germination and early growth of young maize seedlings. Accumulation of the maize malate dehydrogenase isozymes is probably controlled by synthesis as well as degradation instead of activation and inactivation of enzyme moieties. Correlation between the developmental pattern of the scutellar malate dehydrogenase isozymes and the physiological conditions of the young maize seedlings is discussed.


The preparation of several new nylon tube-glucose oxidase derivatives and their incorporation into the "reagentless" automated analysis of glucose. Nylon tube was activated by alkylation with dimethyl sulfate and used for the immobilization of glucose oxidase. Lysine, hexamethylene diamine and polyethylene imine were also attached to activated nylon tube, and these nylon tube-spacer derivatives were reactivated with either glutaraldehyde or ethyl adipimidate for the subsequent coupling of glucose oxidase. The activities of all of the different nylon tube-glucose oxidase derivatives were compared by their incorporation into standard Technicon automated analysis systems. Activities were measured either spectrophotometrically, by following the production of hydrogen peroxide using an acid/KI assay, or polarographically by following the decrease in the oxygen concentration using a flow-through oxygen electrode assembly. The activity and stability of all of the nylon tube-glucose oxidase derivatives was such that their use in the routine estimation of glucose levels was an attractive proposition.


Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. 1. Lipoyl dehydrogenase (NADH: lipoamide oxidoreductase, ED 1.6.4.3) and two asparagusate dehydrogenases from asparagus mitochondria were purified by a series of steps, freezing and thawing, sodium dodecylsulfate extraction, and chromatography on Sephadex G-200 and DEAE-cellulose.  2. Lipoyl dehydrogenase was highly specific for alpha-lipoic acid, which could not be replaced at all by asparagusic acid.  Each of the asparagusate dehydrogenases was capable of reducing both asparagusic and alpha-lipoic acids by using NADH as hydrogen donor.  3. Reduction of alpha-lipoic cid with NADH by lipoyl dehydrogenase was activated by NAD, but that of asparagusic acid by asparagusate dehydrogenase was inactivated by NAD.  4. Lipoyl dehydrogenase and two asparagusate dehydrogenases differed in electrophoretic mobility on polyacrylamide gels.


The role of alkalication in formation and decomposition of myosin-ATP complex. The dependence of kinetic constants Km, V(k2) and k1 of myosin-ATPase on the species and concentration of alkali cations and on temperature was investigated.  The value of Vvaries with the ionic radius of different alkali cations.  The curve has a maximum at 1.33 A at the ionic radius of potassium.  The detailed analysis of the cation dependence of the kinetics of the ATPose reaction shows that both formation and decomposition of the complex are affected by the cation present.


Regulation of phenylalanine ammonia-lyase synthesis by cinnamic acid.  Its implication for the light mediated regulation of the enzyme. 1. There is an increase in the extractable acitivity of phenylalanine ammonia-lyase following excision of hypocotyl segments of dark-grown gherkin seedlings  This increase is prevented by the reaction product, cinnamin acid.  2. Density-labelling experiments show that cinnamic acid affects the rate of enzyme synthesis.  By contrast the regulation of phenylalanine ammonia-lyase activity by light has previously been shown to involve activation of existing inactive enzyme.  3. It is proposed that regulation of synthesis by reaction products represents a mechanism for controlling the size of the pool of phenylalanine ammonia-lyase, and that the activity of this pool is regulated by light.


Metabolism of N-carbobenzoxyl-L-tryptophan by Chromobacterium violaceum. Chromobacterium violaceum (ATCC 12472) metabolizes N-carbobenzoxyl-L-tryptophan into its 2', 3'-dehydro-derivative, and indole-3-propionic acid into indole-3-acrylic acid.  The biotransformation accurs in resting cell incubations, and in growing cultures of the bacterium.  Tryptophan in fermentation media enhances the ability of Chromobacterium violaceum to perform the conversion by an undetermined mechanism.  The amino acid also prevents stored cultures from losing the ability to accomplish the biotransformation.  The reaction apparently requires oxygen, and preliminary experiments suggest that it is catalyzed by a dehydrogenase.


Hyaluronic acid salt--a mechanoelectrical transducer. Hyaluronic acid transduces a very gentle pressure into an electrical potential.  Such pressure, depending on its direction, changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the unpressured salt or changing and increasing the rotation in the opposite direction.  These finding have implications for understanding the function of the cochlear and vestibular fluids, renal function, and the approximation to frictionless motion of normal joints.


Interactions between the macromolecule and the gradient-forming solute in isopycnic sedimentation. Interactions between the macromolecules and the gradient-forming solute in isopycnic sedimentation equilibrium experiments affect the distribution of the macromolecules in a number of ways.  A description is given of the effects that may be expected, and some recently reported results are interpreted in the light of these.  Suggestions are made for the recognition of such interactions, and for their possible effects, both good and bad, on experimental results.


Transferrin in the reticulocyte cytosol. Radioactive iodine-labeled iron-saturated human transferrin was shown to enter the cytosol of rabbit reticulocytes but not erythrocytes, and to be combined therein with a small "carrier" material not identical to the membrane transferrin receptor.


NMR study of -17-O from H2-17-O in human erythrocytes. Human erythrocytes were incubated in a Ringer's solution enriched with 10--18 per cent H2-17-O.  The longitudinal relaxation time (T1) of the -17-O was determined separately in samples of red cell suspensions, packed cells, and supernatant.  The longitudinal relaxation of -17-O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.  The T1 of intracellular -17-O is 4--5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz).  This datum is consistent with tht thesis that hemoglovin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.  The rate constant (kx) for water exchange was calculated to be 60 and 107 s- minus 1 at 25 and at 37 degrees C, respectively.  The apparent activation energy for kx over the temperature range 23--37 degrees C was 8.7 plus or minus 1.0 kcal/mole.


Water in normal muscle and muscle with a tumor. The total water content, the amount of non-freezable water, and the Na-+ and K-+ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T1) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to minus 65 degrees C. Quantitatively calculated T1 values are given. The difference in T1 for the two types of muscle at temperatures above minus 5 degrees C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na-+.


Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion. Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain. The concentration of substance P for half maximal stimulation in rat brain was 1.8-10-minus 7 M. The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E1 and 184% by prostaglandin E2. Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca-2+ above 10-minus 6 M. The chelating agent ethyleneglycol-bis-(beta-aminoethylether)-N, N'-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity. The inhibition by ethyleneglycol-bis-(beta-aminoethylether)-N, N'-tetraacetic acid was completely reversed by increasing concentrations of Ca-2+.


Studies on the relationship of hepatic anion-binding proteins and sulfobromophthalein-glutathione conjugation in normal and phenobarbital-treated rats. The enzyme activity which conjugates sulfobromophthalein with glutathione was separated from rat liver supernate by Sephadex G-75 gel filtration, and assayed by two different methods; paper electrophoresis and spectrophotometry. The enzyme activity was found mainly in the second protein fraction, and less than 5% of the activity in the first or third protein fractions. In vitro mixtures of sulfobromophthalein, [3-H]glutathione and rat liver supernate showed that the major part of [3-H]glutathione was detected in the first and second protein fractions, and the remainder in the third protein fraction. Phenobarbital treatment caused an increase of the enzyme activity, sulfobromophthalein and [3-H]glutathione, in the second protein fraction.


Subcellular fractionation of pig platelets. Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or alpha-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the beta-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16--1.17) having a specific activity 10--100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and beta-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. Beta-N-acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.


Composition of cartilage from lysozyme-deficient rabbits. Costal and auricular cartilage obtained from mutant rabbits exhibiting lysozyme deficiency has been found to be identical to similar tissue from control animals in a variety of biochemical parameters. These data seriously question the putative role of lysozyme as a structural component of cartilage.


Thermally induced conformational changes of tobacco mosaic virus and their protein assemblies. The measurement of the fluorescence intensity of tryptophan residues showed that a reversible transition in the local structure took place between 20 degrees C and 30c in intact virus particles and reconstituted protein assemblies of the ordinary strain and the tomato strain of tobacco mosaic virus. During this transition the overall polymer structure was maintained. In the case of the bean strain of tobacco mosaic virus, however, the fluorescence intensity did not show any transition in the same temperature range. Such a difference between different strains gave some information on the location of tryptophan residues possibly involved in the local structure change. The fluorescence polarization of intact virus particles showed no change in the whole temperature range, but the polarization of the reconstituted protein assembly of the ordinary strain showed a transition at the same temperature as the fluorescence intensity. This suggested a difference in the freedom of the local structure between intact virus particles and reconstituted protein assemblies. Oligomers of these virus proteins were stable up to 45c and above this temperature, began to make an irreversible transition where the secondary structure of the monomer was partially broken but the oligomer structure was retained.


Circular dichroism study of the conformation of ultraviolet-irradiated ribonuclease A. RNAase A irradiated by ultraviolet light at 254 nm shows a linear dependence between loss of activity and destruction of cystine. At least one of the cystine modified forms in irradiated RNAase is catalytically active. Circular dichroism spectra of irradiated RNAase show a marked decrease in ellipticity between 210 nm and 230 nm, an increased ellipticity between 230 nm and 240 nm, and a blue shift of the 210-nm minimum toward 205 nm. These circular dichroism changes indicate a pariial disorganization of the native secondary and tertiary changes with irradiation. The temperature dependency of the circular dichroism shows the irradiated enzyme to be conformationally  less stable to thermal perturbation than native RNAase. Differences in the polypeptide conformations of unirradiated RNAase denatured by heat and sodium dodecylsulfate, and irradiated RNAase treated with heat and sodium dodecylsulfate are discussed.


On the presence of a heat-stable, macromolecular inhibitor of the thrombin-fibrinogen reaction in rat liver microsomes and its separation from prothrombin. By concentrating sonicates from rat liver microsomes containing prothrombin, the activity as measured by the one-stage prothrombin assay (Hjort, P., Rapaport, S. J. and Owren, P. A.(1965), J. Lab. Clin. Med. 46, 89-97) gradually decreased. Nearly a complete loss of prothrombin activity was found in sonicates being concentrated 10-fold. By adsorption of prothrombin on barium citrate and dissolving the precipitate in a solution of sodium citrate. NaCl and EDTA followed by gel filtration on a Sephadex G-50 column, the inhibitory effect on the bioassay of prothrombin disappeared. An inhibitor of the thrombin-fibrinogen reaction could be isolated from the supernatant after adsorption of prothrombin on barium citrate. The inhibitor was excluded from a Sephadex G-50 column equilibrated with 4 M NaCl in veronal buffer (pH 7.4) and was heat stable (70 degrees C for 10 min). No proteolytic or antitrypsin activity could be detected in the inhibitor preparation. The importance of removing the inhibitor of the microsomal sonicates prior to any bioassay of coagulation factors based on the thrombin-fibrinogen reaction is emphasized.


2,3-Dioxo-5-indolinesulfonic acid, a new highly specific reagent for modification of tryptophan in peptides and proteins. A new reagent, 2,3-dioxo-5-indolinesulfonic acid (DISA), has been investigated for its specificity to react with tryptophan and tryptophan residues in peptides and proteins. On reaction of 19 amino acids with 1 M excess of DISA in 0.1 M acetic acid (pH 2.9), considerable (50%) modification of tryptophan was obtained within 50 min and no other amino acid was modified. After reaction for 5 h, only proline showed very slight (7%) modification. On reaction in the presence of 5 M excess of DISA, tryptophan was very rapidly modified. Modification of proline became appreciable in the presence of this molar excess of reagent and cysteine modification, although much smaller, became detectable (12 h, 7%). However, proline modification was completely prevented when this amino acid was engaged in a peptide linkage, even after reaction for 45 h in the presence of 5 or 10 M excess of DISA per proline residue. Reaction of egg albumin with 50 M excess of DISA was entirely specific for tryptophan and showed no modification of proline or cysteine residues. The reagent offers the advantages of stability, easy handling, high water solubility and high specificity. It affords protein and peptide derivatives that are completely water soluble because of the polar nature of the added group. The yellow color (lambda max, 367 nm) of the derivatives offers advantages of easy determination of the extent and location of the modification.


The helical content of zein, a water insoluble protein, in non-aqueous solvents. The conformation of zein, a seed protein from corn, was determined by optical rotary dispersion measurements in a wide variety of nonaqueous solvents. Over a wide range of dielectric constants, in pure and mixed solvent systems, the helical content was independent of dielectric constant. Determination of the helical content of insulin and ribonuclease in several of these solvents indicated a variation in secondary structure comparable of that of zein. Though virtually insoluble in water, zein not only is a globular protein in nonaqueous solutions, but has conformational properites characteristic of more conventionally behaving globular proteins.


Mossbauer spectra of bicarbonate-free ferric-transferrin complex. The bicarbonate-free ferric-transferrin complex was investigated by Mossbauer Spectroscopy under anaerobic conditions. No evidence of specific binding was found. The spectral results indicates that the apparent binding is due to the formation of antiferromagnetic ferric hydroxide polymers which are firmly attached to the transferrin molecule. Specific binding would only occur in the presence of bicarbonate or other low molecular weight chelators.


Characterization of trypsin and elastase from the moose (Alces alces). I. Amino acid composition and specificity towards polypeptides. Trypsin and elastase isolated from the pancreas of the moose (Alces alces), a member of the Cervidae (deer) family, were characterized with respect to their amino acid composition and specificity towards polypeptides. Moose trypsin possessed 234 residues, based on alanine recoveries equal to 16.0 residues, with a molecular weight calculated at 24 476. Moose trypsin readily hydrolysed peptide bonds in which the carbonyl group was contributed by arginine, lysine and S-2-aminoethylcysteine as indicated by the peptides isolated following hydrolysis of the oxidized and the S-aminoethylated B-chain of insulin. Moose elastase possessed 231 residues, based on alanine recoveries equal to 17.0 residues, with a molecular weight calculated as 24 201. The high lysine (9 residues), low arginine (3 residues) content was in contrast to the opposite situation with porcine elastase and the elastase-like, alpha-lytic protease from Sorangium. The hydrolysis of the oxidized B-chain of insulin by moose elastase was similar to that produced by  porcine elastase with major cleavages occurring at Val-12-Glu-13, Ala-14-Leu-15 and Val-18-Cys(O-3H)-19 and minor cleavages occurring at Ser-9-His-10 and Arg-21-Gly-22. The hydrolysis of glucagon with moose elastase produced major cleavages at Thr-7-Ser-8, Ser-11-Lys-12, Val-23-Gln-24 and Leu-26-Met-27. The facile hydrolysis of Arg-17-Arg-18 was also observed and attributed, in part, to trypsin.


The disociation of bovine factor V and isolation of the acitivated form. Bovine Factor V disociated non-enzymatically to 276 000 dalton, whereas, the enzymatic activation produced Factor Va of 213 500 daltons. The disociated form, Factor Vd, was not an enzyme substrate for conversion to Factor Va. Factors V and Va sedimented in a density zone of 1.19 g/ml, and Factor Vd in 1.17 g/ml, in sucrose gradient centrifugation. Only a neutral lipid in a 7.2% yield could be extracted from Factor Va which had been isolated in a 4500-fold purification.


Circular dichroism of erythrocyte membrane glycoproteins. The circular dichroism spectra were obtained for purified equine, human and bovine membrane glycoproteins, which have 40, 55 and 70% carbohydrate, respectively. The spectra in aqueous buffer show similar shapes, maxima and minima but somewhat different peak amplitudes. Analysis of the spectra indicated that the glycoproteins can be pictured as existing primarily in an unordered form in dilute aqueous buffer with small amounts of alpha-helix (13-23%) present. In 2-chloroethanol, a helix-promoting solvent, the amount of alpha-helix is increased to 60-70%. The glycoproteins underwent denaturation in guanidine hydrochloride, although evidence of some residual structure did remain. The spectra of the glycoproteins change relatively little on going from aqueous buffer to dodecylsulfate solutions. Removal of 60% of the sialic acid does not induce significant conformational alterations. The anomalous behavior of the glycoproteins during molecular weight determinations does not appear to be related primarily to conformational restrictions on the polypeptide chain.


Oxygen equilibrium and subunit aggregation of a holothurian hemoglobin. The hemoglobin of the sea cucumber Cucumaria miniata Brandt has a mol. wt of about 36000 in the oxy- form with a s20,w equal to 2.9 and a subunit molecular weight of 18000 by sodium dodecylsulfate gel electrophoresis. This pigment aggregates when deoxygenated to an oligomer with a s20,w equal to 4.7, an aggregation which is reversible upon subsequent oxygenation. The hemoglobin shows a sigmoid binding equilibrium with "n" equal to 1.8 and a decrease in oxygen affinity with an increase in pigment concentration. This  hemoglobin is compared with other hemoglobins showing oxygenation-linked subunit aggregation.


Properties of Ascaris muscle mitochondria. 1. Cytochromes. 1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations. 2. Difference spectra (at 22 degrees C and -196 degrees C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N' ,N'-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c1, c and aa3, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component. 3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150mM KCl, leaving the membrane-bound cytochromes c1, b and aa3 in the KCl residue.


The kinetics and specificity of electron transfer from cytochromes and copper proteins to P700. The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with those from three other c-type cytochromes and azurin, a copper protein resembling plastocyanin. Three different disruptive techniques were used to expose P700; digitonin, Triton X-100 and sonication. The following rate constants were measured at 25 degrees C, pH 7.0, with digitonin-treated chloroplasts: plastocyanin, 8 x 10(7)M(-1) x s(-1); red-algal cytochrome c-553, 1.9 x 10(7)M(-1) x s (-1); Pseudomonas cytochrome c-551, 8 x 10(6)M(-1) x s (-1); azurin, less than or = 3 x 10(5)M(-1) x s (-1); cytochrome f, less than or = 2 x 10(4)M(-1) x s (-1); mammalian cytochrome c, less than or = 2 x 10(4)M(-1) x s (-1). For electron transfer from plastocyanin, the effects of ionic strength, pH and temperature were also studied, and saturation effects found in earlier work were avoided by a full consideration of the various secondary reactions and inclusion of superoxide dismutase. The relative rates are discussed in relation to photosynthetic electron transport.


Electron transport in an in vitro-reconstituted bacterial photophosphorylating system. Photooxidation of endogenous cytochrome(s) c, photoreduction of endogenous chrome(s) b and photobleaching of bacteriochlorophyll have been demonstrated in an in vitro reconstituted system, previously demonstrated to support photophosphorylation. The kinetic responses of these redox reactions to substrate and antimycin A in these particle are characteristic of electron transport processes and stronglysupport the contention that all, or a part of, the oxidative phosphorylation electron transport pathway can be coupled to reaction center, photopigment complex in a manner which supports photophosphorylation. In addition, a succinate-supported light dependent reduction of NAD+ was found.


Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts. IV. The effects of electron-transport conditions on violaxanthin availability. 1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electron-transport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than from cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear flow from reduced N-methyphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity. 2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (greater than or equal to 1 mu M) had no effect on availability, whereas low levels of 3, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50 percent decrease at 1 mu M). Furthermore, availability in 3,3-(3',4'-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone. 3. Violaxanthin availability was zero in the dark and increased in the light to maximum of 67 percent of the total violazanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.


Fluorescent kinetics of chlorophyll in photosystems I and II enriched fractions of spinach. The fluorescent emission kinetics of spinach subchloroplast Photosystems I and II particles have been studied on a picosecond time scale. Using picosecond laser pulses and an optical Kerr gate, the fluorescent decay times are measured to be 60 plus or minus 10 ps, and 200 plus or minus 20 ps for Photosystems I and II, respectively. The quantum yields are calculated to be 0.004 for Photosystem I and 0.013 for Photosystem II. Theory of exciton energy transfer and trapping is applied for the determination of intermolecular potential energyin the photosystems.


Analysis of anaerobic fluorescence decay in Scenedesmus obliquus. With reduction of System II acceptors during dark anaerobic adaptation in Scenedesmus obliquus fluorescence yield rises to a maximum value in two distinct transitions. Subsequent illumination results in a decay of fluorescence yield with the following characteristics: 1. In low intensity light it is independent of temperature and is an expression of light reaction I. 2. In high intestity light it reflects the dark limiting step in the reoxidation mechanism of System II primary acceptors. 3. There is strong inhibition by agents known to block electron transport between the two systems. 4. At light limiting conditions decay kinetics include an initial delay phase and thereafter close to second order behaviour. 5. Following a single brief saturating flash a maximum of 80% quenching is restored and a second flash yields approx. 95% restoration. Comparison with the fluorescence rise in the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea suggests that the decay reflects transfer of one positive charge from System I to the primary System II acceptor with the intermediary carrier pool remaining reduced.


[Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti (author's transl)]. Studies of absorbance related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 mu mol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.


Energy-dependent accumulation of iron by isolated rat liver mitochondria. IV. Relationship to the energy state of the mitochondria. 1. The energy-dependent accumulation of iron by isolated rat liver mitochondria, respiring on endogenous substrates, is strongly dependent on the efficiency of energy coupling in the respiratory chain as measured by respiratory control with ADP and the endogenous energy dissipation. The accumulation reached a saturation level at respiratory control with ADP values (with succinate as the substrate) of approx. 4.0. 2. In the presence of exogenous substrate, the energy-dependent accumulation of iron was markedly reduced, primarily due to binding of iron as carboxylate complexes having less favourable constants than the iron (III)-sucrose complex(es). 3. The effect of added ATP was at least 2-fold, i.e. that of providing energy and that of chelating iron. When the mitochondria respired on endogenous substrate, the energy-dependent accumulation of iron increased at low concentrations of ATP, whereas higher concentrations (greater than 50 mu M) gradually inhibited the uptake. 4. Energization of the mitochondria by the generation of an artificial K-+ gradient across the inner membrane with valinomycin in a K-+-free medium increased the energy-dependent accumulation iron.


Energy-dependent accumulation of iron by isolated rat liver mitochondria. V. Effect of factors controlling respiration and oxidative phosphorylation. 1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria. 2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark T. (1973) Biochim. Biophys. Acta 305, 29-40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c1) and a(+a3). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation. 3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg-2+ is present in the suspending medium. In contrast to Ca-2+, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 mu M the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex. 4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine mucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the P/O ration is slightly diminished.


Mechanism of action of agents which uncouple oxidative phosphorylation: direct correlation between proton-carrying and respiratory-releasing properties using rat liver mitochondria. The proton-carrying properties of uncoupling agents were investigated by measuring passive mitochondrial swelling under conditions where electrogenic proton transport was rate limiting.  The ability of uncoupling agents to transport protons into mitochondria, measured in this way, was compared with respiratory stimulation.  The results show that with the single exception of arsenate, all agents tested which uncouple oxidative phosphorylation demonstrate a very close correlation between release of respiration and proton transport.  These findings are in support of Mitchell's original proposal that uncoupling agents act by promoting electrogenic hydrogen ion transport across the mitochondrial inner membrane.


Circular polarization of fluorescence of chlorophyll in solution and in native structures. Chlorophyll dimers in solution, subchlorplast particles and chloroplasts were investigated by their circular dichroism and circular polarization of their fluorescence, which reflect their optical rotatory power in the ground state and electronically excited state, respectively.  The chlorophyll dimers in fluid solution lose their optical activity upon electronic excitation, reflecting a marked concomitant change in the structure of the dimers.  This change is arrested in a solution of very high viscosity.  The pronounced difference between the circular polarization of the dimers in fluid media and that of subchloroplast particles and chloroplasts indicates that the former are not suitable models for associated chlorophyll in native structures in electronically excited states.  Impairment of the photochemical activity of chloroplasts by heat treatment is accompanied by a reduction of the circular polarization of the fluorescence, which probably reflects a disorganization in structure.  The same extent of circular polarization was observed in the fluorescence of chloroplasts regardless whether the reaction centers are open or closed; thus either the same molecules are emitting in the two cases or, if different molecules emit, they are packed in a similar way.