Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.     

Characterization by cytofluorometry of the different types of adenohypophyseal cells in the rat

The different antehypophysical cell types which synthetize and release somatotroph (GH), corticothroph (ACTH), gonadotroph (LH-FSH) and lactotroph (PRL) hormones were analysed. The experiments were performed on hypophyses from five groups of animals: adult males, 14 days-old female, adult females, gestating adult females and lactating adult females. The cells were analysed by immunofluorescence using flow cytometry. For each of the hormones studied, there was a characteristic spectral distribution of cells. The evolution of cell size and granular content with respect to sex and physiological state of each group was studied by the analysis of diffused light. Small, slightly granular cells represented 50% of the cell population in males and 14 day-old females but only 8% in gestating or lactating females. The study of the cell cycle showed the presence of dividing cells in the population of large, granular cells from gestating and from lactating females. No features of cell division were observed in the population of small, slightly granular cells. This study indicates the potential value of multiparametric analysis in the separation of pure sub-populations of antehypophysial cells.     

Low-temperature electron paramagnetic resonance study of the ferricytochrome c-cardiolipin complex

The electron paramagnetic resonance spectrum of the ferricytochrome c complex with cardiolipin was observed at temperatures below 20 K. For the low-spin iron(III) heme system complexed with the negatively charged lipid, the tetragonal and rhombic ligand field parameters (delta/lambda = 3.58, V/lambda = 1.82) differ significantly from those (delta/lambda = 2.53, V/lambda = 1.49) of the free ferricytochrome c sample. The g values of the complex (gx = 1.54 +/- 0.02, gy = 2.26 +/- 0.01, gz = 3.02 +/- 0.01) are compared to the values for free ferricytochrome c (gx = 1.25 +/- 0.02, gy = 2.25 +/- 0.01, gz = 3.04 +/- 0.01). Spectral alterations are interpreted in terms of the ligand field changes induced within the heme group by association with the negatively charged phosphoglyceride.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

Tissue distribution of a novel neurotensin-degrading metallopeptidase. An immunological approach using monospecific polyclonal antibodies.

A monospecific polyclonal antiserum was raised against a recently purified rat brain neurotensin-degrading metallopeptidase. The purified IgG fraction immunoprecipitated the peptidase and inhibited its proteolytic activity. Western blot analyses revealed that the immune fraction recognizes only one protein in rat brain homogenates, and this corresponds closely to the purified enzyme. The IgG displayed a restricted specificity towards the peptidase from murine origin. In the rat, the neurotensin-degrading enzyme was widely distributed throughout peripheral organs with the noticeable exception of the duodenum. In addition, the peptidase was detected in various cell lines or membrane preparations of neural or extraneural origin in which it had been previously characterized by means of biochemical methods. In light of this widespread distribution, the putative role of the peptidase in the metabolism of neuropeptides is discussed.     

Unusual genetic phenomena associated with Tn5 mutagenesis in Alcaligenes eutrophus strain H1

Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council