Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.     

Functional expression and CNS distribution of a beta-alanine-sensitive neuronal GABA transporter.

The synaptic action of gamma-aminobutyric acid (GABA) is terminated by high affinity, Na(+)-dependent transport processes in both neurons and glia. We have isolated a novel GABA transporter cDNA, GAT-B, which encodes a high affinity (Km = 2.3 microM), Na(+)- and Cl(-)-dependent GABA transport protein that is potently blocked by beta-alanine, a compound generally considered a selective inhibitor of glial transport. However, in situ hybridization studies indicate that GAT-B mRNA is expressed predominantly within neurons. These data indicate that the neuronal-glial distinction of GABA transporters based on inhibitor sensitivities must be reconsidered and suggest a greater diversity of GABA transporters than has been predicted by previous pharmacologic studies.     

Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis.

gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.     

Mesencephalic dopamine modulation of pituitary and central beta-endorphin: relation to food intake regulation

Pituitary and central beta-endorphin have been implicated in the regulation of food intake. It has been suggested that an elevation in hypophyseal beta-endorphin represents the genetic defect in the obese mutant Zucker rat. Both pituitary and central beta-endorphin systems appear to interact with dopamine. We have therefore examined hypophyseal, hypothalamic, and basal forebrain levels of beta-endorphin in the obese Zucker rat, its lean littermate, and lean littermates sustaining neurotoxic lesions of the A10 dopamine cell group in the ventral mesencephalon. The obese mutant exhibits elevated pituitary, but not central, beta-endorphin levels relative to lean littermates. A10 lesions result in a marked increase in both pituitary and hypothalamic beta-endorphin levels, and tend to decrease the amount of the peptide in the basal forebrain. These lesions do not result in either increased food intake or body weight. These data therefore suggest that elevated pituitary beta-endorphin levels do not mediate obesity in the Zucker rat, and also demonstrate that both central and pituitary beta-endorphin are modulated by a dopamine system originating in the ventral mesencephalon.     

Sensitivity of Escherichia coli to proline analogues during osmotic stress and anaerobiosis

L.M. REESE, K.O. CUTLER AND C.E. DEUTCH. 1996. The sensitivity of wild-type Escherichia coli K-12 to a series of proline analogues was determined in cultures containing increasing concentrations of NaCl under both aerobic and anaerobic conditions. The bacteria were most sensitive to L-azetidine-2–carboxylate and L-thiazolidine-4–carboxylate. The minimum inhibitory concentrations for these compounds decreased progressively during osmotic stress, but the bacteria were much more sensitive to these proline analogues under aerobic conditions than during anaerobiosis. The reduced sensitivity under anaerobic conditions did not reflect degradation of the compounds in the culture medium. Since both urine and medullary renal tissue contain relatively low oxygen concentrations, these results raise doubts about the potential use of proline or glycine betaine analogues in treating urinary tract infections.     

Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.     

Escherichia coli delta 1-pyrroline-5-carboxylate reductase: gene sequence, protein overproduction and purification.

The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.     

Arginyl-tRNA-protein transferase in eucaryotic protists

Soluble extracts of $\text{Saccharomyces cerevisiae}$ and $\text{Blastocladiella emersonii}$ were found to catalyze the specific transfer of arginine from a mixture of [¹⁴C] aminoacyl-tRNAs into protein. Arginine transfer was stimulated by bovine serum albumin. Glu-Ala, Asp-Ala and cystinyl-$\text{bis}$-Ala inhibited incorporation into protein, whereas dipeptides with other NH₂-terminal residues linked to alanine did not. These results indicate the presence of an enzyme in eucaryotic protists with the same donor and acceptor specificity as mammalian arginyl-tRNA-protein transferase.