Distribution of sodium-plus-potassium-stimulated adenosine-triphosphatase activity in isolated nerve-ending particles

1. A rapid method for the isolation of nerve-ending particles from brain is described. This involved the centrifugation of the large-granule fraction over a discontinuous density gradient consisting of 3% (w/v) and 13% (w/v) Ficoll dissolved in 0.32m-sucrose. The results of the biochemical as well as morphological identification of nerve-ending particles are given. 2. Approx. 20% of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity originally present in the cerebral grey-matter suspension was recovered in the fraction consisting principally of large nerve-ending particles (approx. 1mu in diameter). The activity of the adenosine triphosphatase/mg. of protein in the nerve-ending fraction approximated to that in the small-granule fraction after the treatment with glycol ether diamine-tetra-acetic acid. The conclusion was drawn that the synaptic structure, supposedly the limiting membrane of the nerve-ending particle, is one of the feasible sites of localization of the (Na(+)+K(+))-stimulated adenosine-triphosphatase activity in cerebral tissues. Adenosine triphosphatase in purified cerebral mitochondria was not stimulated by Na(+). 3. No qualitative differences were found between the (Na(+)+K(+))-stimulated adenosine-triphosphatase activities exhibited by the nerve-ending particles and by the cerebral small-granule fraction with respect to pH-dependence, cation requirements and susceptibility to ouabain.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection.

The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.