Effect of pH, salt, and coupling state on the interaction of ferredoxin with the chloroplast membrane

To determine if the interaction between ferredoxin and ferredoxin:NADP reductase is similar to the interaction between the purified proteins when the ferrodoxin:NADP reductase is membrane bound, the effect of pH, salt, and coupling state on the Km for ferredoxin in NADP reduction by chloroplast membranes has been examined. Increasing pH and salt concentrations as well as uncouplers all resulted in increases in the Km for ferredoxin. The pH and salt effects on the Km are similar to effects observed by others (C. Batie and H. Kamin (1981) J. Biol. Chem. 256, 7756-7763) on the dissociation constant for a complex between the two purified proteins, although the salt effect on the Km appears to be affected by the surface potential of the chloroplast membrane. These results suggest that the interaction between ferredoxin and the membrane-bound ferredoxin:NADP reductase is not greatly different from the interaction which has been characterized between the two purified proteins.     

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.     

Calorimetric studies of flavin-binding proteins: FMN and FAD binding to hen egg riboflavin-binding proteins

Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 10⁵ and 10⁴ were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form.     

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P₂-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP₃), and the α₁-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP₃, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.     

Haem-binding proteins of the rat liver cytosol

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino [³h] laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [³H] haem-labelled mitochondria, [³H] haem-labelled microsomes or with [³H] haemin.These results are discussed with particular reference to ligandin.     

The use of transferred nuclear Overhauser effects in the study of the conformations of small molecules bound to proteins

A novel method is proposed for the study of the conformation in solution of small molecules bound to proteins. In transfer of saturation experiments, irradiation at the frequency of a proton in the bound ligand can result in an intensity change in the signal from a different proton in the free excess ligand via a nuclear Overhauser effect between the two protons in the bound ligand. Approximatel calculations show that the observation of such effects depends upon the close spatial proximity (within about 4.0 Å) of the two protons involved and thus gives useful conformational information. Two examples of this method are given, for the binding of trimethoprim and NADP⁺, respectively, to Lactobacillus casei dihydrofolate reductase.     

Synthesis of affinity-label chelates: a novel synthetic method of coupling ethylenediaminetetraacetic acid to amine functional groups

An affinity-label chelate for the enzyme trypsin was synthesized by a novel synthetic technique which takes advantage of the presence of a dangling carboxylate arm in the [Co(EDTA)Cl]2- complex anion. The dangling carboxylate group was coupled to the amino group of p-aminobenzamidine, an effective inhibitor of trypsin activity, via the carbodiimmide reaction to produce a trypsin affinity label at one end and a strong EDTA-like chelating agent at the other, coupled through an amide bond. The cobalt ion can be removed if desired by reduction with Fe2+ + ascorbate, and alternate metal ions inserted in its place. The reaction is general, and affinity labels which contain amino groups can be easily coupled via this procedure, allowing the introduction of a paramagnetic or fluorescent probe into a protein or nucleotide system. The same method has been used to prepare a highly effective chelating gel which is capable of removing calcium and lanthanide ions from the binding protein parvalbumin.     

Translation of the mRNAs coding for the major hemolymph proteins of Ceratitis capitata in cell-free system: comparison of the translatable mRNA levels to the respective biosynthetic levels of the proteins in the fat body during development

Four major hemolymph polypeptides (ceratitins) with molecular weights between 8.1 X 10(4) and 8.7 X 10(4) daltons have been identified in the fat body of late Ceratitis capitata larvae. Total fat body RNA from late larvae was translated in reticulocyte lysate, and the predominant in vitro translation products were shown to be the ceratitin precursors. The biosynthesis of these proteins during postembryonic development was studied in both tissue culture and cell-free system. Comparison of the biosynthetic patterns obtained in the two systems suggests a linear relationship between messenger concentration and protein synthesis. Three of these polypeptides show a coordinate pattern of synthesis and are immunologically related. After pupation, all four ceratitins are reabsorbed by the fat body where they accumulate.     

Crosslinking of chemically reactive A-U-G analogs to ribosomal proteins within initiation complexes.

A-U-G analogs, either reactive on their 5′ or their 3′ side, were employed in affinity labeling of the ribosomal A-U-G binding site. These experiments have been carried out such that the chemically reactive A-U-G analog became covalently bonded to ribosomal proteins only in the presence of fMet-tRNA_f^Met and initiation factors. Subsequent radioimmunodiffusion of A-U-G-labeled proteins identified proteins IF3, S1, S18, S21 and L11 as being in the neighborhood of the ribosomal codon binding site. A location of reactive sites of these proteins relative to the P or A site bound codon is, however, not clear.The A-U-G labeling results are quantitatively as well as qualitatively very different in the absence or presence of fMet-tRNA_f^Met. It is concluded, therefore, that fMet-tRNA_f^Met directs A-U-G into its final binding site. Streptomycin cannot release fMet-tRNA_f^Met from initiation complexes which contain irreversibly bound 5′- {4-(bromoacetamido)phenylphospho}-adenylyl-(3′–5′)-uridylyl-(3′–5′)-guanosine. This suggests that codon-anticodon interaction between A-U-G and fMet-tRNA_f^Met is still intact in the P site of the ribosome.     

A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome

Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein $\text{L}\text{7}\text{L}\text{12}$) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8.     

Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon

Precise frameshift and nonsense mutations were introduced into the region preceding the galactokinase gene (galK) of Escherichia coli. These mutations after the position at which upstream translation terminates relative to the galK translation initiation signal. Constructions were characterized that allow ribosomes to stop selectively before, within or downstream from the galK initiation signal. The effects of these mutations on galK expression were monitored. Galactokinase levels are highest when upstream translation terminates within the galK initiation region. In contrast, when translation stops either upstream or down stream from the galK start site, galK expression is drastically reduced. These results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon (that is, galT), and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the galK start site. Possible mechanisms and implications of this translational coupling phenomenon are discussed.     

Transplantation of marine sponges to different conditions of light and current

Transplantation was employed to determine how marine sponges grow in different in situ conditions of light and current. The growth of Verongia aerophoba (Schmidt), which contained symbiotic cyanobacteria. was enhanced in light, particularly when sediment was excluded by a clear shield. V. cavernicola Vacelet and Chondrosia reniformis Nardo. which did not contain cyanobacteria, grew preferentially in the shade and were inhibited by light. The growth of Petrosia ficiformis (Poirct) and Chondrilla nucula Schmidt, which may or may not contain cyanobacteria. did not appear to be favoured by either light or shade. The growth of sponges in lower current speeds was considerably reduced ; this reduction was. however, partially reversed in those sponges with cyanobacteria when grown in the light. Presumably symbiotic cyanobacteria are beneficial to sponges growing in the light because they transfer photosynthetically fixed nutrient and shield the underlying tissue. Significant morphological differences were observed in sponges grown under different environmental conditions.     

Second-derivative spectroscopy of proteins: Studies on tyrosyl residues

Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.     

Isolation of two proteins with 9-ketoprostaglandin reductase and NADP-linked 15-hydroxyprostaglandin dehydrogenase activities and studies on their inhibition

Two proteins containing 9-ketoprostaglandin reductase and NADP-linked 15-hydroxyprostaglandin dehydrogenase activities have been isolated. Both of these activities are inhibited by a low molecular weight placental protein and by indomethacin, ethacrynic acid, and furosemide.     

Structural studies of hydroxycitrates and their relevance to certain enzymatic mechanisms.

The crystal structures of the ethylenediamine salts of two diastereoisomeric hydroxycitratesy are described, and their conformations in the solid state are analyzed. In both structures, the HOCCOH torsion angle is approximately 60 ° as found for many tartrates and mesotartrates. The presence of three carboxyl groups and two hydroxyl groups in hydroxycitrates leads to 10 possible types of tridentate metal chelates. Since bacterial citrate lyase and ATP citrate lyase require metal ions, the possible geometries of hydroxycitrate chelation have been compared with those of citrate, and as a result, some information on the geometry of each enzymic active site has been obtained. If the hydroxycitrate binds in the same manner as citrate, the C(3)&;z.sbnd;C(4) bond will be in the correct position to be cleaved. Other modes of binding of hydroxycitrate, if they can be accommodated in the active site of the enzyme, are nonproductive and compete with the citrate-like mode causing inhibition. It is possible, in these alternate modes of binding of hydroxycitrates, for additional binding to side chains in the active site of the enzyme to occur, resulting in extremely potent inhibition.