ABSTRACT
The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.
Subject(s)
Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Proteins/isolation & purification , Endopeptidases/isolation & purification , Lysobacter/enzymology , Peptides/isolation & purification , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Endopeptidases/chemistry , Endopeptidases/immunology , Immunoenzyme Techniques , Peptides/chemistry , Peptides/immunologyABSTRACT
Activity of phosphate PPHO or arabinose PBAD promoter of Escherichia coli has been studied depending on the content of zwitter-ionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or under significant decrease in the content of anionic phospholipids, there is a significant decline of PPHO promoter activity but not PBAD promoter. Since the PPHO promoter belongs to the Pho-regulon--a member of the family of two-component regulatory systems of signal transduction having membrane sensors, the regulation of gene expression by phospholipids is presumed to be realized through a membrane sensor.
Subject(s)
Escherichia coli/metabolism , Membrane Lipids/metabolism , Phospholipids/metabolism , Promoter Regions, Genetic , Base Sequence , DNA, Bacterial , Escherichia coli/genetics , Molecular Sequence Data , Signal TransductionABSTRACT
cDNAs coding for three types of alpha-subunits of GTP-binding proteins Gs and G0 (a short form of alpha s with Asp-Ser in positions 71 and 72, a long form of alpha s with the insertion of 16 amino acid residues instead of Asp-Ser (71-72)--both from bovine brain, and alpha 0 from bovine cerebellum as well as some chimeric alpha s/alpha 0 genes were cloned into a modified pGEM-2 plasmid vector under the control of the SP6 promoter. All the genes were in vitro transcribed and translated, and some functional properties of the resulting proteins were determined, such as adenylyl cyclase activation, ADP-ribosylation with pertussis toxin, limited nucleotide-dependent trypsin proteolysis. Parts of the alpha s polypeptide chain necessary for the activation of adenylyl cyclase were mapped. The alpha s domain interacting with adenylyl cyclase is formed by the alpha s polypeptide chain fragments 235-294 and 337-356 (numbering as of the alpha s long form).
