NADH-dependent glutamate synthase and nitrogen metabolism in Neurospora crassa

The GDH (NADPH) mutant strain $\text{am-1}$ of $\text{N. crassa}$ has sizable pools of glutamine and glutamate under ammonium-limited conditions for which requires an elevated glutamine synthetase activity. Glutamine in the pre$\text{s}$ ence of 2-oxoglutarate, stimulated nicotinamide nucleotide oxidation by crude and purified extracts of the $\text{am-1}$ strain and led to a reductant dependent formation of two molecules of glutamate. Aminooxyacetate did not have any effect on the reaction, whereas azaserine inhibited it completely. It is concluded that in $\text{N. crassa}$ glutamine synthetase and glutamate synthase are responsible for the assimilation of low ammonium concentrations.     

The p-cymene pathway in PseudomonasputidaPL: Isolation of a dihydrodiol accumulated by a mutant

A mutant strain (PL pT $\text{11}\text{43}$) of $\text{Pseudomonas}\text{putida}$ PL, has been isolated for its inability to growth with $\text{p}$-cymene as carbon source. The mutant oxidizes $\text{p}\text{-cymene}$ (and $\text{p}$-cumate) to a compound (λmax 293 nm) which is readily converted to 3-hydroxy-$\text{p}$-cumate by acid. 4-Trifluoromethylbenzoate is oxidized by the mutant to an acid-stable intermediate (λmax 277nm) that has been crystallized. The spectral properties (u.v., i.r., NMR and mass) of this metabolite are consistent with those expected for a 2,3-dihydro-2,3-dihydroxy derivative of 4-trifluoromethylbenzoate. Further support of this structure was provided by elemental analysis and the properties of two derivatives of the metabolite, 4-trifluoromethyl-3-hydroxybenzoate and an acetonide formed with 2,2-dimethoxypropane. The stability of a product obtained by treatment of the dihydrodiol metabolite with triacetylosmate indicates that it is the $\text{cis}\text{-isomer}$.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.     

The influence of dna A and dna C mutations on the initiation of plasmid DNA replication

The dependence of the replication of several plasmids on the chromosome-determined initiation products, $\text{dna}$ A and $\text{dna}$ C, has been studied. The initiation of the replication of $\text{Col}$ E₁ DNA requires the chromosomal $\text{dna}$ A product. In contrast two de-repressed transfer factors ($\text{R 1 drd 16}$ and $\text{Hly}$₁₅₂) seem to determine a corresponding plasmid-specific factor. The $\text{dna}$ C-product is necessary for the ordered initation of all plasmids studied. The addition of low concentrations of chloramphenicol leads to a relaxed replication of $\text{Col}$ E₁ DNA at the restrictive temperature in $\text{dna}$ A-mutants, but not in $\text{dna}$ C-mutants.     

Membrane lipid modifications: Biosynthesis and identification of phosphatidyl-N-methyl-N-isopropylethanolamine in rat liver microsomes

In previous studies on the modification of polar head groups of membrane phospholipids with the unnatural base analog, $\text{N-}\text{isopropylethanolamine}$, we reported an unidentified phospholipid in addition to $\text{phosphatidyl}\text{-N-}\text{isopropylethanolamine}$ in the various membrane fractions of rat liver. The structure of this phospholipid has now been identified as $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$ by nuclear magnetic resonance spectroscopy, and by chromatographic and enzymic analysis. In addition, we found that when rats were injected intraperitoneally with the $\text{N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$, 19% of teh liver microsomal phospholipid was $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$.     

Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus

The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism $\text{Streptomyces hygroscopicus}$, has been cloned in the $\text{Streptomyces}$ vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of $\text{Streptomyces hygroscopicus}$ DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the $\text{Streptomyces}$ vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Invitro evaluation of granulocyte differentiation of diffusion chamber inocula

The semi-soft agar colony assay permits an $\text{in}$$\text{vitro}$ analysis of committed myeloid stem cell (CFU-c) proliferation capacities. In this paper this procedure has been used in combination with prior diffusion chamber culturing to determine the effect of host influences upon this committed stem cell population. This “double-seeding” procedure of first culturing bone marrow cells in diffusion chambers and then re-seeding them in agar furnishes data suggesting a relationship between $\text{in}$$\text{vivo}$ diffusion chamber transitional lymphocytes and $\text{in}$$\text{vitro}$ CFU-c seeding capacities. Diffusion chamber culturing offers a means of monitoring granulopoiesis and selects for enrichment of stem cell numbers. Detection and quantification of diffusion chamber stem cell enrichment is easily assessed by seeding chamber contents into the agar colony assay.     

A new prenyltransferase from Micrococcuslysodeikticus

A new prenyltransferase which catalyzes the synthesis of geranyl pyrophosphate as the only product from dimethylallyl pyrophosphate and isopentenyl pyrophosphate has been separated from other known prenyltransferases from $\text{Micrococcus}\text{lysodeikticus}$. This enzyme fraction is also capable of synthesizing all-$\text{trans}$ geranylgeranyl pyrophosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate though it lacks ability to synthesize farnesyl pyrophosphate.     

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.     

Analysis of the inhibitor binding to the nucleoside site of phosphorylase b

The binding of inhibitors to site I of rabbit muscle phsphorylase $\text{b}$ has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both $\text{Δ}\text{H}$ and $\text{Δ}\text{S}$ of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase $\text{b}$ is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive $\text{T}$ conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active $\text{R}$ conformation, whereas non-exclusive binding of the activator was observed when caffeine was added.     

Effect of E. coli endotoxin on the structure-function of fatty acid synthetase lipoprotein

The lipoprotein structure of the fatty acid synthetase complex from Ceratitis capitata has been used as a model to $\text{val}\text{i}$ date the claim that phospholipids from membranes assume a $\text{signif}\text{i}$ cant role in the cell-endotoxin interactions. The enzyme-complex was exposed to a ¹⁴C-lipopolysaccharide preparation and the $\text{inte}\text{r}$ action was followed by a) circular dichroism spectra, b) enzyme activity and c) gel filtration chromatography. It should be $\text{emph}\text{a}$ sized that the E. coli endotoxin modifies all these properties of the enzyme complex and that a model involving phospholipids and phase transitions has been proposed to account for these $\text{intera}\text{c}$ tions.     

Limulus anti-LPS factor: An anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system

Exposure of $\text{Limulus}$ amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of $\text{Tachypleus}\text{tridentatus}$ and $\text{Limulus}\text{polyphemus}$. The principle purified partially from $\text{Tachypleus}$ amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the $\text{Limulus}$ coagulation system was discussed.     

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.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

Release and purification of micrococcus lysodeikticus ATPase from membrane extracted with n-butanol

The ATPase associated with the membranes of Micrococcus ysodeikticus has been released into the aqueous phase (i.e. solubilized) by extracting the membranes with $\text{n-}\text{butanol}$ in a two-phase system modified from the procedure of Maddy, A.H. (164) Biochim. Biophys. Acta 88, 448–449. A procedure for the release and purification of the ATPase from the membranes extracted with $\text{n-}\text{butanol}$ is described as an alternate method to that previously used for the shock-wash ATPase. Upon extracting the membrane suspensions with $\text{n-}\text{butanol}$ the soluble ATPase released into the buffer phase no longer exhibits stimulation by trypsin in contrast to the shock-wash type of ATPase. As shown by Salton, M. R. J. and Schor, M. T. (1972) Biochem. Biophys. Res. Commun. 49, 350–357, the shock-wash ATPase possesses associated protein(s) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis whereas these are absent from the purified ATPase released by the $\text{n-}\text{butanol}$ method. The specific activities of the purified ATPase released by the two methods were generally similar, the $\text{n-}\text{butanol}$ type being consistently somewhat higher.