Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.     

Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells?

Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G₁ cells or metabolically blocked G₁/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle.     

Pulse fluorimetry study in polarized light of DNA-ethidium bromide complexes

In previous works, a quantitative analysis of the fluorescence anisotropy decay, based on a comparison of the experimental measurements with a Monte Carlo simulation of the excitation energy migration, has been shown to provide the value of the unwinding angle of the DNA helix, induced by an ethidium bromide (E.B.) molecule intercalation. In the present work some of the characteristics of the model used in the computation are reexamined: namely the influence of the direction of the E.B. electronic moment, and the influence of the dye distribution along the DNA helix are studied. The computations are compared with experimental results obtained with new experiments performed with calf thymus and micrococcus lysodeikticus DNA-E.B. complexes. It is found that the difference in base composition of these DNA does not influence the fluorescence properties of their E.B. complexes. Our study confirms the validity of the dye distribution obtained with the single adjacent excluded site principle. Reasonable values of the unwinding angle are obtained by assuming that the transition moment direction lies along the great axis of the E.B. molecule. The value of this unwinding angle is compared with other values proposed in the literature.     

The tibial thread-hairs ofAcheta domesticus L. (Saltatoria,Gryllidae)

Summary The ultrastructure of the thread-like hairs (sensilla) on the tibia of the front leg ofAcheta domesticus (Gryllidae) Saltatoria was examined by serial sectioning. The presence of a tubular body indicates that these sensilla are mechanosensitive; electrophysiological measurements also confirmed this. The opposing forces on the articulating apparatus of single hairs and the sensitivity of the single receptor cell were measured after deflection of the hair in different directions. The articulating apparatus is characterized by three cuticular elements: a joint membrane, suspension fibers, and a socket septum. These elements form the basis for a structural bilateral symmetry along whose plane of symmetry the direction line of both the minimum receptor sensitivity and the minimum opposing forces lie. The tubular body embedded in the tip of the socket septum is attached to the base of the hair shaft. The hair provides the leverage for displacing the tubular body and the socket septum limits the extent to which it may be laterally displaced.These investigations have been supported by the Deutsche Forschungsgemeinschaft     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

Synthesis and characterization of 2′-azidoaminopterin as a potential photoaffinity label for folate-utilizing enzymes

A photoreactive analog of aminopterin, 2′-azidoaminopterin (VI), was synthesized and evaluated as a potential inhibitor and photoaffinity label of folate-utilizing enzymes. The compound was tightly bound to dihydrofolate reductase (DHFR) from escherichia coli (MB 1428) with K₁ equal to 3 × 10^?11M and to the enzyme from mouse (S-180) cells with K₁ approximately equal to 2 × 10^?10M. Dissociation constants measured by equilibrium dialysis using radioactive 2′-azidoaminopterin gave a value of K_D = 3.2 × 10^?9M for the bacterial enzyme. The presence of NADPH enhanced the affinity by more than an order of magnitude. Azidoaminopterin is also an inhibitor of thymidylate synthetase from Lactobacillus casei, competitive with methylene-tetrahydrofolate (K_i 7 × 10^?7M). Photolysis of the radioactive inhibitor in complex with DHFR from E. coli led to approximately 3% covalent incorporation of label into protein. The greater part of this attachment was nonspecific as shown by the lack of protection in the presence of methotrexate. Thymidylate synthetase from L. casei was not significantly inactivated upon photolysis in the presence of the inhibitor and deoxyuridylate. Model studies showed that photoreaction of the inhibitor led to covalent linkages with thiol, lysyl amino groups, and the hydroxyl groups of alcohols. Azidoaminopterin may be useful in labeling other enzymes of folate metabolism, although a minor photoproduct reacts nonspecifically with many proteins. The antifolate can be photoconjugated to polylysine as well as to proteins. The polylysine conjugates inhibit DHFR. Difference spectrum analysis of the photoproducts from the irradiation of the DHFR I complex indicates that water reacts efficiently with the enzyme-bound nitrene and must therefore have access to at least part of the bound p-aminobenzoyl group. This analysis suggests that azide analogs of protein ligands may be useful as reporter groups in probing the hydrophobicity of binding sites.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distances between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformational change, leading to a fluorescence quenching of the tryptophanyl emission.     

A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH,which yields activated resins,free of interfering nitrogen derivatives

A new approach for the reaction of Sepharose with cyanogen bromide is described, using triethylamine as a “cyano-transfer” reagent. An optimized procedure for activation at neutral pH was developed. This procedure requires only about 5% of the usual amount of cyanogen bromide. Activated resins are free of imidocarbonates and carbamates, containing only active cyanate esters. Extremely high coupling capacities (75 μmol ligand/g wet Sepharose 4B) can be obtained using this method.