bluuebunny/crossref_metadata_2025 huggingface dataset:
Separation of hydrogenase-catalyzed hydrogen-evolution system from electron-donating system by means of enzymic electric cell technique
Authors: Yagi Tatsuhiko
Published: May 2006

An enzymic electric cell was constructed in which the anode was composed of a zinc plate inserted in aqueous NH
4
Cl solution and the cathode was composed of an electrode made of a glassy carbon stick inserted in a reaction mixture containing cytochrome
c
3
hydrogenase (H
2
:ferricytochrome
c
3
oxidoreductase, EC 1.12.2.1) and methylviologen under an atmosphere of N
2
. When the circuit was closed, the electric cell formulated as [Formula: see text] was composed, and hydrogenase-catalyzed H
2
-evolution was observed in the cathode of the cell with concomitant flow of an electric current. Thus, the hydrogenase-catalyzed reaction and the electron-donating reaction proceeded at different parts of the cell. This enables us to protect the hydrogenase from the byproducts of the electron-donating system, which might be hazardous to the enzyme.
Amine-catalyzed hydrolyses of cyclodextrin cinnamates
Authors: Komiyama Makoto , Bender Myron L.
Published: May 2006

Hydrolyses of β-cyclodextrin cinnamate (βCDC) and α-cyclodextrin cinnamate were catalyzed by amines such as 1,4-diazabicyclo(2.2.2)octane, triethylamine, quinuclidine, piperidine, diisobutylamine, and
n
-butylamine. The rate constant of hydrolyses of the βCDC-amine complexes follows the order: 1,4-diazabicyclo(2.2.2)octane >
n
-butylamine > quinuclidine > piperidine > triethylamine ≫ diisobutylamine. The ratio of the catalytic rate constant for the βCDC/1,4-diazabicyclo(2.2.2)octane complex to the spontaneous rate constant for βCDC is about 6-fold and is almost independent of pH below pH 11.5; but, it then drastically increases with pH above pH 11.5, up to 57-fold at pH 13.6 which is much higher than previous attempts. The pH-rate constant profile and isotope effect with deuterium oxide solvent indicate that 1,4-diazabicyclo(2.2.2)octane, included in βCDC, assists the catalytic nucleophilic attack by hydroxide ion toward the carbonyl carbon of βCDC. Acceleration of deacylation of acyl-cyclodextrins, by amines, has made the cyclodextrin-catalyzed hydrolysis of esters an even better model of hydrolytic enzyme reactions than those developed previously.
Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor.
Authors: Low B W , Preston H S , Sato A , Rosen L S , Searl J E , Rudko A D , Richardson J S
Published: May 2006

The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor. The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparallel beta-pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the sevent invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue.
Purification and characterization of initiation factor IF-E3 from rabbit reticulocytes.
Authors: Benne R , Hershey J W
Published: May 2006

Initiation factor IF-E3 from rabbit reticulocytes was isolated from a high salf extract of ribosomes prepared according to the procedure of Schreier and Staehelin (J. Mol9 Biol, 73, 329-349, 1973). The factor was highly purified from the crude extract by ammonium sulfate fractionation, sucrose gradient centrifugation, salf gradient elution from DEAE-cellulose and phosphocellulose columns, and glycerol gradient centrifugation. IF-E3 stimulated cell-free protein synthesis dependent on an exogenous globin mRNA fraction 4- to 5-fold. The factor under nondenaturing conditions behaved as a large multipolypeptide complex, but was separated into 11 major protein components by two-dimensional polyacrylamide gel electrophoresis with urea and sodium dodecyl sulfate. The stoichiometry and molecular weights (range: 28,000-140,000) of the IF-E3 proteins were determined. None of the components corresponded to ribosomal proteins found in high salt-washed ribosomes. 14CH3-IF-E3 was prepared by reductive alkylation without detectable loss of its initiation factor activity, and bound stoichiometrically to 40S ribosomal subunits, but not to 60S or 80S ribosomes. 14CH3-IF-E3 isolated from the 40S complex contained only nine of the 11 original protein components.
Correlation of degradative rates of proteins with a parameter calculated from amino acid composition and subunit size.
Authors: Momany F A , Aguanno J J , Larrabee A R
Published: May 2006

A parameter is developed which relates the amino acid composition and subunit size of a protein to the degradative rate in vivo. This parameter was calculated for 11 rat liver proteins and a plot versus the half-lives of these proteins is linear and has a coefficient of correlation of -0.96. Evidence is presented which suggests that the density of excess acidic amino acids on the surface of the protein is the most important factor in determining differential turnover.
Synthesis of a 19-residue peptide with alamethicin-like activity.
Authors: Gisin B F , Kobayashi S , Hall J E
Published: May 2006

This paper describes the chemical synthesis of a compound with voltage-gating characteristics similar to those observed in nerve membranes. For alamethicin (ALA), a natural antibiotic that induces such properties in lipid bilayer membranes, there are two proposed structures, one a cyclic and the other an open chain peptide. The open chain sequence (ALA-o) proposed by Martin and Williams [(1976) Biochem. J. 153, 181-190] was synthesized by stepwise solid-phase condensation of four fragments prepared by solid-phase synthesis. The product, purified to homogeneity, was not identical with the main component of natural ALA. Nevertheless, in lipid bilayer membranes the exponential dependence of conductance on voltage and the dependence of conductance on a high power of the peptide concentration were qualitatively similar for ALA-o and for natural ALA. Like ALA, ALA-o showed the characteristics of a channel-former, although the single-channel conductances were less well defined for the synthetic compound. This work establishes that a cyclic structure is not a necessary condition for a peptide to induce voltage-dependent conductances in membranes and that ALA-o possesses all the structural elements required for such an activity.
Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.
Authors: Chacko S , Conti M A , Adelstein R S
Published: May 2006

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.
Stable conformations of aliphatic disulfides: influence of 1,4 interactions involving sulfur atoms.
Authors: Van Wart H E , Scheraga H A
Published: May 2006

The present state of knowledge of the stable conformations of the XCCSSCCX fragment (in which X is any saturated group) of aliphatic disulfides is examined. In particular, the evidence for the existence of attractive 1,4 interactions between CH groups and S atoms across C-S bonds and their influence on the potential function for rotation about C-S bonds is discussed. The effects of similar attractive interactions between NH groups and S atoms on the potential function for rotation about C-C bonds also are considered. It is concluded that weak attractive interactions between CH groups and S atoms are capable of stabilizing rotamers with unusually low (about 30 degrees) values of the SS-CC dihedral angle.
Inhibition of prostaglandin endoperoxide synthetase by thiol analogues of prostaglandin.
Authors: Ohki S , Ogino N , Yamamoto S , Hayaishi O , Yamamoto H
Published: May 2006

A variety of thiol compounds inhibited the enzymatic bis-oxygenation of 8,11,14-eicosatrienoic acid to prostaglandin G1, as examined with a purified preparation of prostaglandin endoperoxide synthetase (prostaglandin synthase; 8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase; EC 1.14.99.1) from bovine vesicular gland. The hydroperoxide cleavage of prostaglandin G1 producing prostaglandin H1 was not affected by these thiol compounds. Several prostaglandin analogues with a thiol group (9,11-dihydroxy-15S- or 15R-mercaptoprosta-5,13-dienoic acid, 1-mercapto-9,11,15-trihydroxyprosta-5,13-diene, and 1-mercapto-9-oxo-11,15-dihydroxyprosta-5,13-diene) were most potent inhibitors, showing almost complete inhibition at concentrations on the order of 1 muM. Other thiol compounds, such as 2,3-dimercaptopropanol, dithiotherreitol, and dihydrolipoic acid, were also inhibitory but were much less effective. The inhibition, as examined with 9,11-dihydroxy-15S-mercaptoprosta-5,13-dienoic acid and 2,3-dimercaptopropanol, was noncompetitive.
Experimental test of the vibronically coupled tunneling description of biological electron transfer.
Authors: Potasek M J , Hopfield J J
Published: May 2006

Evidence for the constructs central to vibronically coupled electron transfer has been obtained. Our experiments show the existence of a weak (f congruent to 10(-6)) charge-transfer absorption band in the near infrared for the bound donor-acceptor complex, cytochrome c-Fe(CN)6. Such a charge-transfer band had been predicted from the theory of such transfers. The experimental method, using a form of excitation modulation spectroscopy, measures only the optical absorption that induces charge transfer between the donor and the acceptor (and does not detect other absorptions) and allows the study of charge-transfer bands whose absorbances are small compared to the sample absorbance. The energy position and oscillator strength of the band agree with the general predictions of this vibronically coupled tunneling theory. We suggest that, in this system at room temperature, the electron transfer can be described by this tunneling theory. This model system result gives credence to the short electron transfer distances the theory has predicted for biological electron transfers.
Chromosome mapping of the genes that control differentiation and malignancy in myeloid leukemic cells.
Authors: Azumi J I , Sachs L
Published: May 2006

The chromosome banding pattern has been analyzed in clones of mouse myeloid leukemic cells that differ in their ability to be induced to differentiate by the protein inducer MGI (macrophage and granulocyte inducer). None of the clones had a completely normal diploid banding pattern. The clones studied were either MGI+ (that can be induced to form Fc and C3 rosettes), a stage in the differentiation of myeloid cells, or MGI- (that cannot be induced to form these rosettes). All six cultured clones of MGI- cells from myeloid leukemias independently produced in six separate animals showed a loss of a piece of one chromosome 2 and this abnormal chromosome was maintained in leukemias derived from the cultured cells. This loss was not found in MGI+ clones or lymphoid leukemias. Five MGI+ mutants, derived from an MGI- clone with a loss of a piece of one chromosome 2, one normal chromosome 12, and two translocated chromosomes 12, maintained the abnormal chromosome 2 but lost either the one normal or one of these translocated chromosome 12. These results indicate that chromosomes 2 and 12 carry genes that control the differentiation of myeloid leukemic cells and that inducibility by MGI is controlled by the balance between these genes. We suggest that these chromosomes also carry genes that control the malignancy of these cells.
Rates of aggregation, loss of anchorage dependence, and tumorigenicity of cultured cells.
Authors: Wright T C , Ukena T E , Campbell R , Karnovsky M J
Published: May 2006

The net rate of spontaneous aggregation of cells suspended with EDTA was measured for various cell types including spontaneous transformants and cells transformed with DNA and RNA viruses. The anchorage dependence as determined by growth in methyl cellulose and the tumorigenicity in vivo were also determined. All cells that had lost their anchorage dependency and were tumorigenic showed a high net rate of spontaneous adhesion. A31 was the only nontransformed cell line to have a high net rate of adhesion. The net rate of spontaneous aggregation of cells is a quick and reliable index of tumorigenicity and offers a new approach to understanding the mechanisms of cell surface changes associated with transformation.
Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line.
Authors: Boon T , Kellermann O
Published: May 2006

Cells from the malignant teratocarcinoma line PCC4.azal were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Fifty-five clones were isolated from the surviving cells. Twelve clones are unable to form tumors in the syngeneic 129/Sv mice. However, these "tum-" clones form tumors as readily as the original cells when they are injected into irradiated mice. Moreover, they stimulate the production of immune memory cells, which protect the injected animals and confer resistance by adoptive transfer. The tum- clones are therefore unable to generate tumors in syngeneic mice because they elicit an immune rejection response.
In vitro evidence of cell-mediated immunity after exposure of mice to both live and inactivated rabies virus.
Authors: Wiktor T J , Doherty P C , Koprowski H
Published: May 2006

Mice exposed to live or beta-propiolactone-inactivated rabies virus generated a strong, specific cell-mediated cytotoxic response which was generally maximal 6 days after inoculation. Release of 51Cr was apparently a function of immune thymus-derived lymphocytes (T cells) because it was abrogated by prior incubation of spleen cells with anti-thymus antiserum and complement but was undiminished by passage of spleen cells through nylon-wool columns. Cytotoxicity was always maximal for interactions in which thymus-derived cells and targets shared H-2 genes but, unlike the situation found in other assays of this type, considerable lysis of allogeneic, virus-infected target cells may also occur. Perhaps the most significant finding from these experiments is that an inactivated virus has been shown to stimulate a potent cytotoxic thymus-derived cell response. Manipulation of this experimental model may allow analysis of the antigens required for stimulation of cell-mediated immunity. A more practical consequence may be the development of more rational protocols for postexposure vaccination against rabies. Prior treatment of mice with antirabies antibody severely depressed the generation of cell-mediated immunity.
Further study of the effect of enzyme-enzyme interactions on steady-state enzyme kinetics.
Authors: Hill T L
Published: May 2006

This paper continues an earlier one [Hill, T.L. (1977) Proc. Natl . Acad. Sci. USA 74, 3632-3632] and presents further introductory examples. Most attention is devoted to a closed linear chain of two-state enzyme molecules with nearest-neighbor interactions. The one-dimensional Ising theory can be used here. The Bragg-Williams (mean field) approximation is introduced to deal with a one-, two-, or three-dimensional lattice of enzyme molecules, at steady state, with an arbitrary kinetic diagram. The behavior of the flux in a phase transition is noted. Finally, a treatment is given for the first effect (second "viral" coefficient) of interactions on the flux in a dilute solution of two-state enzyme molecules.
Local interactions in bends of proteins.
Authors: Zimmerman S S , Scheraga H A
Published: May 2006

Calculated probabilities of bend formation in 47 amino acid sequences of N-acetyl-N'-methylamide dipeptides, determined from a statistical mechanical analysis using empirical conformational energies, were compared with the observed fraction of bends formed in the same 47 dipeptide sequences in the x-ray structures of 20 globular proteins. Agreement between the calculated and observed fraction of bends was found for 26 dipeptides, suggesting that, for those particular dipeptide sequences, local interactions dominate over long-range interactions in determining conformational preference. Seven dipeptide sequences, all of which contained a Gly residue, had a significantly higher calculated than observed bend preference, indicating the strong influence of long-range and/or solvent interactions in those sequences. Of the 14 sequences for which the calculated was significantly less than the observed bend fraction, 13 dipeptide sequences contained at least one polar residue (Ser, Asn, or Asp) and/or an aromatic residue (Phe or Tyr), suggesting that solvent effects may play an important role in dictating the conformation in these sequences. The analysis of dipeptide sequences in the twenty globular proteins also indicated that the 4 leads to 1 hydrogen bond is not a dominant factor in stabilizing bends in proteins, and that most dipeptide sequences are capable of forming several types of bend conformations.
Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin
Authors: Haire Robert N. , Hedlund Bo E.
Published: May 2006

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of sodium chloride in the concentration range from 0.02 to 3.0 M indicate that the oxygen affinity decreases up to about 1 M salt and then begins to increase. The isotherms obtained in the range from 0.02 to 0.6 M sodium chloride, at 37° and pH 7.4, have been analyzed in terms of changes in Gibbs free energy of heme ligation, resulting from the differential interaction between the chloride ion and the two forms of hemoglobin. The maximal theoretical change in Gibbs free energy that chloride ion can exert on the oxygen binding of hemoglobin amounts to 4.9 ± 0.2 kcal/mol (21 ± 0.8 kJ/mol) of hemoglobin tetramer. A plot of the logarithm of oxygen concentration at half saturation versus the logarithm of the chloride concentration has a slope of 0.40, suggesting 1.6 apparent chloride sites per hemoglobin tetramer. Because the interaction between chloride and hemoglobin is dependent on pH, the apparent thermodynamic linkage between chloride and oxygen binding will also include the salt dependence of the Bohr effect at pH 7.4. The fractional change in Gibbs free energy, measured as a function of the chloride concentration, can be approximated by the binding isotherm between a protein and a ligand, using an association constant of 11 M
-1
. Thus, if the number of oxygen-linked chloride sites is more than one per hemoglobin tetramer, these sites must be considered independent.
Use of solvent cavity area and number of packed solvent molecules around a solute in regard to hydrocarbon solubilities and hydrophobic interactions
Authors: Hermann Robert B.
Published: May 2006

Two currently used methods of calculating the area of contact between a solute molecule and the solvent are examined and some important differences are pointed out. Some pitfalls and uncertainties in the application of the concept of molecular surface area to the quantitative estimation of unitary free energies of solution are discussed.
Resonance coherent anti-Stokes Raman scattering spectra of fluorescent biological chromophores: Vibrational evidence for hydrogen bonding of flavin to glucose oxidase and for rapid solvent exchange
Authors: Dutta Prabir K. , Nestor James R. , Spiro Thomas G.
Published: May 2006

Coherent anti-Stokes Raman scattering (CARS) spectra are reported for flavin adenine dinucleotide (FAD) and glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4), in resonance with the 450 nm flavin absorption band. Several isoalloxazine ring modes were observed (1635, 1584, 1507, 1416, and 1359 cm
-1
). A 12 cm
-1
increase in one component of the 1359 cm
-1
band upon binding to glucose oxidase was attributed to hydrogen bonding of the N3 proton to a protein acceptor. This interpretation is consistent with deuteration results. Smaller decreases (5-7 cm
-1
) on binding were observed for the 1635 and 1416 cm
-1
modes (and also a 1297 cm
-1
mode of deuterated FAD) and were attributed to environmental effects. Deuteration of bound FAD was observed within 5 min of mixing glucose oxidase with D
2
O, demonstrating ready access to solvent. Lorentzian CARS peaks were observed with ω
as
at the peak of the 450 nm absorption band, a condition which corresponds to maximum resonance enhancement if the peak corresponds to the envelope of 0-1 vibronic transitions. If ω
1
was tuned to the peak, then dispersion lineshapes were observed, reflecting a loss of Raman enhancement relative to the electronic background.
Unfolding and refolding occur much faster for a proline-free proteins than for most proline-containing proteins.
Authors: Brandts J F , Brennan M , Lung-Nan Lin
Published: May 2006

The kinetics for unfolding and refolding of a parvalbumin (band 5) have been examined as a function of pH near the transition region, using stopped-flow techniques. This protein is rather unusual in that it has no proline residues, and therefore serves as a good example to test the hypothesis that the rate-limiting step seen in denaturation reactions is due to the cis-trans isomerization of proline peptide bonds in the denatured state. The kinetics for parvalbumin unfolding and refolding are complex, with the data being resolvable into two fast phases at 25 degrees. The slower of the two phases seen for the parvalbumin is about 100 to 500 times faster than the slow phase seen for proline-containing proteins under the same conditions! These results argue strongly in support of the proline isomerization hypothesis. It is also suggested that the slower phase seen for parvalbumin and the second-slowest phase seen for proline-containing proteins might be due to the cis-trans isomerization of peptide bonds of non-proline residues.
Structure of a promoter for T7 RNA polymerase.
Authors: Oakley J L , Coleman J E
Published: May 2006

We have determined the nucleotide sequence of a Hpa II restriction fragment of the phage T7 DNA containing a promoter for the phage-specified RNA polymerase. (Hpa II is a restriction endonuclease from Haemophilus parainfluenzae.) Mapping of the Hpa II restriction fragments on the T7 genome shows this promoter to be the second of tandem promoters separated by approximately 170 base pairs that begin transcription by the T7 RNA polymerase at approximately 15% of the genome. Features of the sequence involved in recognition by the T7 RNA polymerase are discussed and include the following region of hyphenated 2-fold symmetry (boxed regions are related through a 2-fold axis of symmetry at the center of the sequence shown). (See article). This sequence includes the initiation site, since the message transcribed from this fragment begins pppG-G-G-A. Combination of our results with work of others has permitted this fragment to be mapped at the junction of T7 genes 1 and 1.1. The RNA transcribed from this fragment begins within gene 1 and contains the RNase III cleavage site that lies between genes 1 and 1.1. This sequence is compared to other processing sites in T7 early message.
Interactions of a photoaffinity analog of GTP with the proteins of microtubules.
Authors: Geahlen R L , Haley B E
Published: May 2006

Tubulin dimers isolated from brain contain two GTP binding sites, a nonexchangeable site and an exchangeable site. To localize the exchangeable site, we used a photoaffinity analog of GTP, 8-azidoguanosine triphosphate (8-N3GTP), which supports tubulin polymerization in the absence of activating light. Photolysis of tubulin polymerized in the presence of 0.01 to 0.1 mM [beta, gamma-32P]8-N3GTP resulted in covalent incorporation of radioactivity only onto the beta monomer. Photolysis with 8-N3GTP also prevented any further repolymerization of the tubulin whereas like treatment in the presence of GTP had no effect. Preincubation of tubulin with GTP prevented photo-incorporation of [beta, gamma-32P]8-N3GTP whereas preincubation with ATP did not.
Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cells.
Authors: Schwarz R I , Bissell M J
Published: May 2006

In an adequate environment, primary avian tendon cells are capable of retaining both the full expression of differentiated function and a correct morphological orientation for 1 week in culture. At high density and in the presence of ascorbate, they are fully stabilized in that they devote 25-30% of their total protein synthesis to collagen, a level comparable to that in tendon cells in ovo. However, either at low density or in medium without ascorbate, they synthesize collagen at only a third of this level. If plated on a collagen matrix, these cells will orient themselves in a manner similar to that of tendon cells in vivo. Furthermore, they are capable of fully modulating the percentage of collagen synthesis upon addition or removal of ascorbate and serum. The variation in the percentage of collagen produced is a result of alterations in collagen synthesis rather than of changes in total protein synthesis or hydroxylation of proline in collagen. Primary avian tendon cells, therefore, provide a suitable model for understanding the stability of the differentiated state, the mechanism of action of ascorbate, and the regulation of collagen biosynthesis.
Recessive dystrophic epidermolysis bullosa: evidence for an altered collagenase in fibroblast cultures.
Authors: Bauer E A
Published: May 2006

Collagenase has been implicated in the pathogenesis of blister formation in recessive dystrophic epidermolysis bullosa. In order to examine whether aberrations in this enzyme are important in the disease, fibroblast cultures from two patients were used to compare the properties of the collagenases from the mutant cells with those from control fibroblast lines. Purified procollagenase preparations from the mutant fibroblasts were significantly more thermolabile at low Ca2+ concentration than control enzymes. They also showed a decrease in affinity for Ca2+, a cofactor required both for enzyme activity and thermal stability. In addition, the collagenase from each mutant line displayed diminished specific activity, expressed as activity per unit of immunoreactive protein, with a mean value of 39% of control for one patient's enzyme and 16% for the other. The data support the postulate that, in these two patients, the altered collagenase is the result of a structural gene mutation, a defect in the post-translational modification of the enzyme, or a mutation in a gene regulating the normal degradation of collagenase.
Oxygen intermediates and mixed valence states of cytochrome oxidase: infrared absorption difference spectra of compounds A, B, and C of cytochrome oxidase and oxygen.
Authors: Chance B , Leigh J S
Published: May 2006

A study of the near-infrared absorption spectra of three oxygen compounds of membrane-bound cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase; EC 1.9.3.1) shows that the formation of compound A (oxycytochrome oxidase) causes no significant infrared absorbance changes at -103 degrees. At -64 degrees, the formation of compound C from the mixed-valence state of the oxidase leads to increased absorption at 740-750 nm. The formation of compound B at -84 degrees from the fully reduced state of the oxidase causes increased absorption at 790-800 nm. Further oxidation of cytochrome oxidase results in increased infrared absorption at 820-830 nm at -60 degrees. The position of the infrared absorption band in compound C thus depends at least upon the oxidation-reduction state of heme a and its associated copper atom. Compound C contains two types of oxidized (cupric) copper; that associated with heme a is initially oxidized, and that associated with heme a3 is oxidized as a second step in the reaction with oxygen. Compound C exhibits a unique intense absorption band at 606-609 nm that is tentatively assigned to a charge transfer interaction between heme a3 in the reduced state and its associated copper in the oxidized state, with heme a and its associated copper in the oxidized state.
Specific neural and adrenal medullary antigens detected by antisera to clonal PC12 pheochromocytoma cells.
Authors: Lee V , Shelanski M L , Greene L A
Published: May 2006

Antisera were prepared against a clonal line (PC12) of rat pheochromocytoma cells that were grown in either the presence or the absence of nerve growth factor (NGF). PC12 cells respond to NGF by growing neurites and express many differentiated properties of adrenal chromaffin cells and sympathetic neurons. The antisera, after absorption with rat liver, kidney, spleen, and thymus, reacted in the micro-complement fixation and indirect immunofluorescence assays with PC12 cells (both NGF-treated and untreated), brain, adrenal medulla, and superior cervical ganglia, but not with a variety of other tissues. Cross-absorption of the antisera with brain and adrenal medulla indicated the presence of components that were specific to brain, adrenal medulla, and PC12 cells. Brain-specific and adrenal-medulla-specific antigens were also detected in other species, such as rabbit, cat, and mouse. No differences could be detected in the specificity of antisera raised against either NGF-treated or untreated PC12 cells. Indirect immunofluorescence on live cells also detected cell surface antigens on both cell bodies and processes. These results suggest that such antisera may be used to detect, localize, and identify specific neural and adrenal medullary antigens.
Roles of ecdysone in
Drosophila
development
Authors: Garen Alan , Kauvar Lawrence , Lepesant Jean-Antoine
Published: May 2006

A temperature-sensitive lethal mutant of
Drosophila melanogaster
called
ecd
1
becomes deficient in ecdysone, as measured by a radioimmunoassay, when there is a shift in temperature from 20° to 29° at various stages of development. Associated with the ecdysone deficiency at 29°, there are abnormalities in larval and imaginal development and the adult functions. When the shift occurs early in third-instar stage, the mutant larvae grow to full size but fail to pupariate, and instead remain living larvae for as long as 3 weeks. These larvae, which have only about 5% as much ecdysone as the wild-type at the time of pupariation, can be induced to pupariate at 29° by ecdysone in their food, indicating that the pupariation block results from an ecdysone deficiency. A shift to 29° later in the third-instar stage does not prevent pupariation of the mutant, but the imaginal discs fail to complete differentiation, although the discs can differentiate at 29° after transplantation to the normal environment of a wild-type host. A shift to 29° early in the first-instar stage blocks a subsequent rise in ecdysone titer and results in extensive developmental defects. Mutant adults become sterile at 29°, and the ecdysone titer in the females concomitantly decreases to 13% of the wild-type value. Mutant larval ovaries transplanted to wild-type female hosts continue to develop and produce competent eggs at 20°, but when the adult hosts are put at 29° the transplanted ovaries become sterile, suggesting that the ecdysone needed for female fertility is synthesized autonomously by ovarian tissue. In contrast to these effects of a shift to 29° during larval, pupal, and adult stages, there is a normal increase in ecdysone titer and normal development in mutant embryos grown at 29°. The insensitivity of the embryo to the
ecd
1
mutation might be due to a maternal contribution of components needed for ecdysone synthesis during the embryonic stage.
13
C nuclear magnetic resonance study of five- and six-coordinated carbon in nonclassical organometallic compounds: Dimeric trialkyl-, tricyclopropyl-, and triarylaluminums and some
nido
and
closo
carboranes
Authors: Olah George A. , Prakash G. K. Surya , Liang Gao , Henold Kenneth L. , Haigh Gary B.
Published: May 2006

A
13
C nuclear magnetic resonance spectroscopic study of dimeric trimethyl-, triethyl-, tricyclopropyl-, and triarylaluminums is reported. The five-coordinated bridging carbons are found consistently more shielded than the terminal carbons, in accordance with the increased p-character of the former. The nature of bridging two-electron three-centered Al—C—Al bonds is discussed.
13
C nuclear magnetic resonance shifts of several
nido
and
closo
carboranes containing five and six coordinated carbons and their
13
C-
1
H spin-spin coupling constants were also obtained. The relationship between the carbon chemical shifts and coordination number of the carbon atom is discussed. There is approximately a 20- to 40-ppm shielding of the
13
C chemical shifts of five- and six-coordinated carbons, compared with those of four valent carbons, with a simultaneous general
increase
of J
C-H
coupling constants.
Light-regulated permeability of rhodopsin:egg phosphatidylcholine recombinant membranes.
Authors: O'Brien D F , Zumbulyadis N , Michaels F M , Ott R A
Published: May 2006

Purified rhodopsin was incorporated into phospholipid bilayers of egg phosphatidylcholine to give recombinant membrane vesicles, which were examined by proton and phosphorus nuclear magnetic resonance spectroscopy. Increased rhodopsin content in the membranes appears to progressively inhibit the molecular motions of the methyl, methylene, and phosphate groups of the phospholipid molecules. This indicates that regions of the rhodopsin molecule interact in a manner that affects the phospholipids from the aqueous interface to the bilayer midline. In the dark, the recombinant vesicles were sealed to europium, manganese, or cobalt ions. Light exposure allowed rapid equilibration of Mn2+ and Co2+, and somewhat slower equilibration of Eu3+ across the membrane. Light changed the membrane permeability, and the gradient in chemical potential resulted in a net ion movement across the rhodopsin:phospholipid recombinant membrane. The results suggest rhodopsin is a transmembrane protein.
Biosynthesis of the covalently linked diglyceride in murein lipoprotein of Escherichia coli.
Authors: Chattopadhyay P K , Wu H C
Published: May 2006

Biosynthesis of the diglyceride moiety of murein lipoprotein in Escherichia coli was studied by pulse-labeling with [2-3H]glycerol and subsequent chase. The evidence strongly suggests that the precursor of the glycerol moiety in lipoprotein is one of the major phospholipid species in E. coli. Studies of biosynthesis of lipoprotein in cerulenin-treated cells indicated that the nonacylated glycerol moiety of phosphatidylglycerol is the donor for the formation of a thioether linkage in the glycerylcysteine residue of the lipoprotein. This is supported by the observation that carbon 1 rather than carbon 3 of sn-glycerol is involved in this thioether linkage. We propose that the biosynthesis of lipoprotein proceeds as follows: apolipoprotein + phosphatidylglycerol or acyl phosphatidylglycerol leads to lipoprotein + phosphatidic acid.
Identification of transmembrane bridging proteins in the plasma membrane of cultured mouse L cells.
Authors: Evans R M , Fink L M
Published: May 2006

Studies were carried out to identify transmembrane bridging proteins in the plasma membrane of mouse L-929 cells. Cells grown in suspension culture were 125I-labeled by lactoperoxidase and allowed to ingest latex particles to produce inside-out membrane phagosome preparations. Phagosomes were isolated and the inner membrane surface was labeled with N-(5'-aminopentyl)-5-dimethylamino-1-naphthalenesulfonamide (dansylcadavarine) by a transglutaminase-catalyzed reaction. The phagosome membrane proteins were solubilized and dansylcadavarine-labeled proteins were isolated by anti-dansyl immunoadsorbent affinity chromatography. Dansylcadavarine-labeled proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography for the presence of 125I-labeled material. By this technique, two iodinated proteins with molecular weights of approximately 50,000 and 80,000 appear to be selectively retained by the anti-dansyl immunoadsorbent, suggesting that these proteins span the plasma membrane.
Angiotensin stimulation of bovine adrenocortical cell growth.
Authors: Gill G N , Ill C R , Simonian M H
Published: May 2006

Factors controlling proliferation of adrenocortical cells have been studied in monolayer cultures of bovine adrenocortical cells. Angiotensin II stimulated cell proliferation and [3H]thymidine incorporation into DNA with a half-maximal effective concentration of 0.96 +/- 0.27 nM. Similar sensitivity to angiotensin III with reduced sensitivity to angiotensin I and tetradecapeptide renin substrate was observed. Although sensitivity to angiotensin II was equivalent to that for fibroblast growth factor (1.5 nM half-maximal effective concentration), maximal effects of angiotensin were less than for fibroblast growth factor and serum. High concentrations of insulin (1-10 micrometer) also stimulated [3H]thymidine incorporation into DNA and cell proliferation. [Sar1,Ile5,Ile8]Angiotensin II, a competitive antagonist of angiotensin II, blocked angiotensin II stimulation of DNA synthesis but did not affect fibroblast growth factor and insulin stimulation of DNA synthesis. Corticotropin (ACTH) blocked the stimulatory effects of both angiotensin II and fibroblast growth factor. The dose-response curves for angiotensin II stimulation of steroidogenesis were similar to those for stimulation of [3H]thymidine incorporation into DNA. Among the seven cell types examined, only adrenocortical cells responded to angiotension II with stimulation of DNA synthesis.
Liposome--lymphocyte interaction: saturable sites for transfer and intracellular release of liposome contents.
Authors: Blumenthal R , Weinstein J N , Sharrow S O , Henkart P
Published: May 2006

The water-soluble dye 6-carboxyfluorescein was trapped in the internal aqueous compartments of small sonicated dioleoyl lecithin vesicles and used to assess the kinetics of transfer of vesicle contents to human lymphocytes. By using flow microfluorometry, the initial rate of dye transfer to the cells was measured as a function of the concentration of vesicles in the external medium. The rate of transfer consists of at least two components, one of which saturates at high vesicle concentration and the other of which does not saturate in the range of concentrations explored. The saturable component was competitively inhibited by vesicles not containing dye. Both the saturable and nonsaturable components of transfer were inhibited by fetal calf serum or bovine serum albumin but neither component was affected by bovine IgG, choline chloride, or heparin. Pretreatment of the lymphocytes with trypsin or Pronase had no effect on either component. The saturable component can be interpreted in terms of a two-step process in which vesicles bind reversely to sites on the cell surface, and dye is then transferred into the cell from the vesicle-site complex.
Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.
Authors: Green J P , Johnson C L , Weinstein H , Maayani S
Published: May 2006

D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.
Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor.
Authors: Burgermeister W , Catterall W A , Witkop B
Published: May 2006

Dihydroisohistrionicotoxin inhibits acetylcholine receptor-dependent 22Na+ uptake of cultured chick muscle cells with a KI of 0.2 micrometer. The inhibition is noncompetitive with respect to agonists. The toxin enhances desensitization of the receptor by agonists which is accompanied by a 10-fold increase in receptor affinity for agonists. Dihydroisohistrionicotoxin increases the affinity of the desensitized form of the receptor for agonists but not antagonists. The results suggest that dihydroisohistrionicotoxin inhibits the acetylcholine receptor by causing an increase in the affinity of the desensitized form of the receptor for agonists and thereby stabilizing the desensitized state.
Functional expression of cloned yeast DNA in Escherichia coli.
Authors: Ratzkin B , Carbon J
Published: May 2006

A collection of hybrid circular DNAs was constructed in vitro using the poly(dA-dT) "connector" method: each hybrid circle contained one molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 X 10(6) daltons. This DNA preparation was used to transform E. coli cells, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids. Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome. Various hybrid ColE1-yeast DNA plasmids capable of complementing E. coli auxotrophic mutations were selected from this population. Plasmid pYeleu 10 complements several different point or deletion mutations in the E. coli or S. typhimurium leuB gene (beta-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12, and pYeleu17 are specific suppressors of the leuB6 mutation in E. coli C600. Plasmid pYehis2 complements a deletion in the E. coli hisB gene (imidazole glycerol phosphate dehydratase). Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon.
A monomeric form of pyruvate kinase in human pyruvate kinase deficiency.
Authors: Adachi K , Ghory P K , Asakura T , Schwartz E
Published: May 2006

A mutant pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human erythrocytes which is easily separated into monomers and dimers by gel chromatography is described. Tht mutant enzyme shows almost the same pH optimum and thermostability as normal enzyme, but has a decreased stability on shaking with air, a decreased Km for phosphoenolpyruvate and a loss of allosteric properties. The apparent Km values for phosphoenolpyruvate of tetramers and monomers were the same. The tetrameric enzyme was slightly activated by fructose-1,6-diphosphate but the monomeric form was not. The tetrameric enzyme was found to dissociate spontaneously to dimeric and monomeric forms.
Homology among acid proteases: comparison of crystal structures at 3A resolution of acid proteases from Rhizopus chinensis and Endothia parasitica.
Authors: Subramanian E , Swan I D , Liu M , Davies D R , Jenkins J A , Tickle I J , Blundell T L
Published: May 2006

The molecular structures of two fungal acid proteases at 3 A resolution have been compared, and found to have similar secondary and tertiary folding. These enzymes are bilobal and have a pronounced cleft between the two lobes. This cleft has been identified as the active site region from inhibitor binding studies. The results of the comparison are discussed in terms of homology among the acid proteases in general.
Surface localization of sites of reduction of nitroxide spin-labeled molecules in mitochondria.
Authors: Quintanilha A T , Packer L
Published: May 2006

The relative rates of reduction of several spin-labeled molecules that partition differently across the hy-drophobic-interface of inner membranes from rat liver mitochondria were investigated. Spin labels localized either deep in the hydrophobic region or in the aqueous phase are only slowly reduced; however a spin-labeled analogue of the cationic detergent cetyltrimethylammonium bromide that partitions at the interface is rapidly reduced by coupled electron transport. Chemical studies on the reduction and oxidation of the spin label show that loss of signal is due to reduction and not destruction of the label. No evidence was found for flip-flop of the label in submitochondrial preparations. Spin reduction of respiring mitochondria, mitoplasts, or inverted submitochondrial preparations is inhibited by rotenone but is relatively insensitive to antimycin A and KCN. Because the midpoint potentials of the spin labels were found to be similar to that of ubiquinone, it is concluded that reducing equivalents of mitochondrial electron transport from this region of the chain are channeled to either membrane interface.
Derepression of ATP sulfurylase by the sulfate analogs molybdate and selenate in cultured tobacco cells.
Authors: Reuveny Z
Published: May 2006

Molybdate and selenate are structural analogs of sulfate that inhibit synthesis of adenosine 5'-phosphosulfate by ATP sulfurylase (sulfate adenylyltransferase, ATP:sulfate adenylyltransferase, EC 2.7.7.4) in crude extracts of tobacco XD cells. Both of these anions derepress ATP sulfurylase in cells growing on sulfate, but not in cells growing on L-cysteine. However, the two anions appear to derepress by different mechanisms. Molybdate caused derepression only at concentrations that were in excess over sulfate and were sufficient to inhibit growth and protein accumulation, indicating that the derepression resulted from sulfur starvation. Selenate caused derepression at one-tenth the concentration of sulfate, a concentration of selenate that was subtoxic, while toxic levels of selenate produced far less derepression. The susceptibility of the tobacco cells to selenate toxicity was high under conditions of sulfur nutrition that derepress ATP sulfurylase, and low under conditions that repress ATP sulfurylase, in agreement with the idea that selenate acts via a functional sulfate assimilation pathway. Since it is known that selenate is incorporated into analogs of sulfur compounds, it is proposed that the tobacco cells synthesize the seleno-analog of the end product of the sulfate pathway responsible for repression, and the seleno-analog antagonizes the normal end product in the repression mechanism, the net result being derepression of ATP sulfurylase by selenate.