Structural features and functional importance of metzincin metalloproteinases

Structural features of metzincin metalloendopeptidases, their physiological role in a cell, and their potential use in medicine are discussed in this article. The authors published their own results of investigations of the new extracellular Bacillius pumilus metalloendopeptidase that exhibited a unique combination of characteristics of both astacin and adamalysin metzincin families.     

Properties of the Bacillus pumilus Glutamyl endopeptidase at different growth stages of its recombinant strain

The Bacillus pumilus 3–19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The glutamyl endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations.     

Construction and characterization of the Bacillus strain with the inactivated phytase gene

The Bacillus subtilis dphy strain with the inactivated phytase gene was obtained. The phytase genes in bacilli displayed high homology; the respective enzymes belonged to the class of β-propeller phosphatases. Physiological and morphological features of the recombinant strain were determined and its sporulation was studied. The level of biomass accumulation was characterized under different conditions of bacterial growth: at different values of pH, temperature, and medium salinity. It is concluded that phytases may participate in stress response formation.     

Association of variable <Emphasis Type="Italic">rs1801282</Emphasis> locus of <Emphasis Type="Italic">PPARG2</Emphasis> gene with diabetic nephropathy

The association of the variable rs1801282 locus of the PPARG2 gene (peroxisome proliferator-activated receptor gamma) with type 2 diabetes mellitus and its complications was analyzed in inhabitants of the Republic of Bashkortostan. The genotype frequencies of the variable rs1801282 locus of the PPARG2 gene did not significantly differ in groups of healthy persons and patients with type 2 diabetes in all three considered inheritance models (codominant, dominant, and recessive). At the same time, it was demonstrated that the risk of one of the diabetic complications, i.e., diabetic nephropathy, was associated with the variable rs1801282 locus of the PPARG2 gene. Diabetic nephropathy was more common in patients with the C/C genotype (62.7%) compared to the C/G and G/G genotypes (37.5%), P = 0.036. The G allele is protective in regard to diabetic nephropathy (OR = 0.36) in patients with type 2 diabetes mellitus.     

The central nucleus of the amygdala: cytoarchitechtonics, neuronal organization, and histophysiology

Neurologists' attention is attracted by amygdala's central nucleus which takes part in formation of stress response and adaptive behaviour in animals. The data on cytoarchitectonics, neuronal organization and histophysiology of this important centre are based on current knowledge of its subnuclear organization.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Isolation and Characterization of Glutamyl Endopeptidase 2 from Bacillus intermedius 3-19

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.     

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase.     

Effect of culture media components on accumulation of glutaminyl endopeptidase in culture liquid of Bacillus intermedius 3-19

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid of Bacillus intermedius 3-19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, while inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca2+, Mg2+, Co2+) increased the production of glutamyl endopeptidase, but others (Zn2+, Fe2+, Cu2+) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase by B. intermedius 3-19 was elaborated.