The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.     

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of $\text{Bacillus}\text{subtilis}$ grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and $\text{NH}{}_4{}^{\mathrm{+}}$. An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.     

Gene m3 mediated efflux of intracellular leucine from bacteriophage P22 infected Salmonella typhimurium

Infection of $\text{Salmonella}\text{typhimurium}$ with the $\text{m}{}_3$ mutant of bacteriophage P22 leads to a rapid and severe efflux of intracellular leucine. The superinfection exclusion ($\text{sie}$) genes of P22 interfere with the function of $\text{m}{}_3$ gene, the product(s) of which is speculated to be an internal protein of phage P22.     

Elasnin,a new human granulocyte elastase inhibitor produced by a strain of Streptomyces

Elasnin, a new human granulocyte elastase inhibitor, has been isolated from $\text{Streptomyces}\text{noboritoensis}$ KM-2753. Elasnin is a neutral, lipophilic colorless and viscous oil ($\text{n}{}_{\mathrm{D}}{}^{17}\text{=1.4983}$, $\text{[α]}{}_{\mathrm{D}}{}^{18}\text{?0.9°}$, λ_max^EtOH 291 nm (ε, 7760)). The molecular formula was C₂₄H₄₀O₄ (M.W.: 392) as determined by its elemental analysis and mass spectrum. Elasnin inhibits markedly human granulocyte elastase, but is almost ineffective for pancreatic elastase, trypsin, chymotrypsin, thermolysin and papain.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Lipid-protein interactions in membrane models fluorescence polarization study of cytochrome b5-phospholipids complexes

According to previous authors, cytochrome $\text{b}{}_5$, when extracted from bovine liver by a detergent method, is called cytochrome $\text{d-b}{}_5$. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome $\text{t-b}{}_5$.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, $\text{R}$, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome $\text{d-b}{}_5$, whereas $\text{R}$ does not change for cytochrome $\text{c}$ and cytochrome $\text{t-b}{}_5$. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome $\text{d-b}{}_5$ occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome $\text{d-b}{}_5$, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

Electron transport components from methanogenic bacteria: The ferredoxin from Methanosarcinabarkeri (strain Fusaro)

A ferredoxin has been isolated from the methanogenic organism $\text{Methanosarcina}\text{barkeri}$ (strain Fusaro). The protein appears to be constituted by two identical subunits of molecular weight approx. 6000 daltons. The UV-visible spectrum of the protein is characterized by two broad absorption peaks centered at 410 and 300 nm and an absorbance ratio $\text{A}{}_{410}\text{A}{}_{300}\text{= 0.8}$. The molar extinction coefficients at 410 and 300 nm are 36,500 and 45,625 M^?1 cm^?1, respectively. The amino acid compsition of $\text{M.}\text{barkeri}$ ferredoxin shows a preponderance of acidic residues and lacks five amino acids. The protein contains 8 cysteine residues and approx. 7 iron atoms and 7–8 acid-labile sulfide groups per molecule which are indicative of the presence of two iron-sulfur clusters in the molecule. The N-terminal sequence shows a high degree of homology with the sequences of ferredoxins from $\text{Clostridium}\text{pasteurianum}\text{,}\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{africanus}\text{.}\text{M.}\text{barkeri}$ ferredoxin functions as an electron carrier in the pyruvate dehydrogenase system. Its possible role in a variety of electron transfer reactions is discussed.     

Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda

A family of plasmid cloning vectors has been constructed that make use of the leftward promoter ($\text{P}{}_{\mathrm{<mtext>L</mtext>}}$) of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ is fully repressed at low temperature by a thermolabile repressor product of the $\text{λc}\text{I}\text{1857}$ gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ control.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

A NMR study of the ionization of fatty acids,fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles

The ionization of fatty acids, fatty amines and $\text{N-}\text{acylamino}$ acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with ¹³C and titrated by using NMR spectroscopy. The apparent p$\text{K}{}_{\mathrm{a}}$ of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p$\text{K}{}_{\mathrm{a}}$ values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p$\text{K}{}_{\mathrm{a}}$ value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p$\text{K}{}_{\mathrm{a}}$ is essentially determined by the low polarity of the lipidic matrix.     

Studies on compartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hapatocytes

The existence of metabolically distinct pools of $\text{S-}\text{adenosyl-L-methionine}$ in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-¹⁴C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-³H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the ³H and ¹⁴C label disappearance in $\text{S-}\text{adenosyl-L-methionine}$ during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of $\text{S-}\text{adenosyl-L-methionine}$. In both yeast and hepatocytes, the ${}^3\text{H}{}^{14}\text{C}$ ratio in $\text{S-}\text{adenosyl-L-methionine}$ decreased sharply. The individual ³H and ¹⁴C decrease in $\text{S-}\text{adenosyl-L-methionine}$ showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct $\text{S-}\text{adenosyl-L-methionine}$ pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The $\text{S-}\text{adenosyl-L-methionine}$ pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel ³H and ¹⁴C decreases in $\text{S-}\text{adenosyl-L-methionine}$.     

Restoration of pool function type behavior to succinate dehydrogenase having latent reconstitutive activity in ubiquinol-cytochrome c reductase complex

Mitochondrial ubiquinol-cytochrome $\text{c}$ reductase complex contains small amounts of succinate dehydrogenase. Estimates from electrophoresis indicate there is one dehydrogenase per eight complexes. This dehydrogenase transfers electrons to the $\text{b}$-$\text{c}{}_1$ complex poorly, as judged by low succinate-ubiquinone and succinate-cytochrome $\text{c}$ reductase activities. Electron transfer to the $\text{b}$-$\text{c}{}_1$ complex is restored by reconstitution of the complex with phospholipid. This phospholipid dependent restoration of electron transfer indicates that either reconstitutive activity of the dehydrogenase is preserved under conditions where electron transfer is absent, or that addition of phospholipid allows one dehydrogenase to transfer electrons to multiple $\text{b}$-$\text{c}{}_1$ complexes.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramylpentapeptide translocase

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by $\text{phospho}\text{-N-}\text{acetylmuramyl}$ (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}$ derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower ($\text{T}{}_1\text{= 16–22°}\text{C}$) and upper ($\text{T}{}_{\mathrm{<mtext>h</mtext>}}\text{= 30°}\text{C}$) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme.