High level production of a peptide hormone analogue [Leu13]motilin inE. coli

Summary A peptide hormone analogue [Leu¹³]motilin has been produced in high yield by recombinant DNA techniques. The peptide was expressed from a multicopied [Leu¹³]motilin gene fused to a salmon growth hormone gene fragment. The monomeric [Leu¹³]motilin was obtained by treatment of the fusion protein with cyanogen bromide, carboxypeptidase A and B. [Leu¹³]motilin showed the equivalent biological activity to that of the natural form.     

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.     

Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy

The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Hydration of clupeine in solution

Spin-lattice relaxation times, T1, of H2(17) O at 25 degrees were measured for aqueous solutions of clupeine and its constituent amino acids, which are serine, threonine, proline and arginine. The dynamic hydration numbers, nDHN, of clupeine and amino acids were determined from a concentration dependence of T1. The coordination numbers nh, and the rotational correlation times, tau ch, of water molecules around clupeine and amino acids were estimated and compared with that of pure water. The tau ch/tau co of clupeine was 1.85 and close to that of arginine. The experimental value of nDHN of clupeine was in good agreement with that calculated from the nDHN values of the constituent amino acids. This means that the clupeine molecule has a random conformation in solution.     

Stable isotope dilution method for the determination of serum glucose using discharge-assisted thermospray liquid chromatography/mass spectrometry

A discharge-assisted thermospray liquid chromatography/mass spectrometric method for the determination of serum glucose was studied. Isotope dilution technique was used with uniformly labelled (13C6) glucose as an internal standard. Successful liquid chromatography/mass spectrometry was achieved by post-column addition of aqueous ammonium acetate to the mobile phase. Quantification was performed by measuring the peak intensity ratios of the unlabelled and labelled [M + NH4]+ ions. Analytical results using the National Institute of Standards and Technology standard reference material serum showed satisfactory agreement with the certified value, and a relative standard deviation of about 1% was obtained.