muramidase and methylamine

Cellular methylamines are osmolytes (low molecular weight organic compounds) believed to offset the urea's harmful effects on the stability and function of proteins in mammalian kidney and marine invertebrates. Although urea and methylamines are found at 2:1 molar ratio in tissues, their opposing effects on protein structure and function have been questioned on several grounds including failure to counteraction or partial counteraction. Here we investigated the possible involvement of cellular salt, NaCl, in urea-methylamine counteraction on protein stability and function. We found that NaCl mediates methylamine counteracting system from no or partial counteraction to complete counteraction of urea's effect on protein stability and function. These conclusions were drawn from the systematic thermodynamic stability and functional activity measurements of lysozyme and RNase-A. Our results revealed that salts might be involved in protein interaction with charged osmolytes and hence in the urea-methylamine counteraction.

Topics: Enzyme Activation; Hydrogen-Ion Concentration; Methylamines; Muramidase; Protein Stability; Proteins; Ribonuclease, Pancreatic; Salts; Thermodynamics; Urea

Human kidney cells are under constant urea stress due to its urine concentrating mechanism. It is believed that the deleterious effect of urea is counteracted by methylamine osmolytes (glycine betaine and glycerophosphocholine) present in kidney cells. A question arises: Do the stabilizing osmolytes, non-methylamines (myo-inositol, sorbitol and taurine) present in the kidney cells also counteract the deleterious effects of urea? To answer this question, we have measured structure, thermodynamic stability (ΔG D (o)) and functional activity parameters (K m and k cat) of different model proteins in the presence of various concentrations of urea and each non-methylamine osmolyte alone and in combination. We observed that (i) for each protein myo-inositol provides perfect counteraction at 1∶2 ([myo-inositol]:[urea]) ratio, (ii) any concentration of sorbitol fails to refold urea denatured proteins if it is six times less than that of urea, and (iii) taurine regulates perfect counteraction in a protein specific manner; 1.5∶2.0, 1.2∶2.0 and 1.0∶2.0 ([taurine]:[urea]) ratios for RNase-A, lysozyme and α-lactalbumin, respectively.

Topics: Enzyme Stability; Humans; Hydrogen-Ion Concentration; Inositol; Kidney; Lactalbumin; Methylamines; Muramidase; Osmolar Concentration; Protein Denaturation; Protein Structure, Secondary; Ribonuclease, Pancreatic; Sorbitol; Taurine; Urea

Kidney cells of animals including human and marine invertebrates contain high amount of the protein denaturant, urea. Methylamine osmolytes are generally believed to offset the harmful effects of urea on proteins in vitro and in vivo. In this study we have investigated the possibility of glycine to counteract the effects of urea on three proteins by measuring thermodynamic stability, ΔGD° and functional activity parameters (K(m) and k(cat)). We discovered that glycine does not counteract the effects of urea in terms of both protein stability and functional activity. We also observed that the glycine alone is compatible with enzymes function and increases protein stability in terms of T(m) (midpoint of thermal denaturation) to a great extent. Our study indicates that a most probable reason for the absence of a stabilizing osmolyte, glycine in the urea-rich cells is due to the fact that this osmolyte is non-protective to macromolecules against the hostile effects of urea, and hence is not chosen by evolutionary selection pressure.

Topics: Animals; Glycine; Humans; Lactalbumin; Methylamines; Muramidase; Osmosis; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Ribonuclease, Pancreatic; Thermodynamics; Urea

T cells recognize foreign proteins as peptides bound to self molecules encoded by the major histocompatibility complex (MHC). The kinetics of interaction between purified class II MHC molecules and peptides is unusual, in that the rate of association is very slow, but once formed, the complexes are extremely stable. This raises the question of how the antigen-presenting cell provides a sufficient number of free MHC binding sites to ensure T cell immunity. We present results suggesting that an exchange of peptide in MHC binding sites may take place under physiological conditions.

Topics: Ammonium Chloride; Animals; Antigen-Presenting Cells; Cell Line; Chloroquine; Fixatives; Glutaral; Histocompatibility Antigens Class II; Hydrogen-Ion Concentration; Interleukin-2; Kinetics; Methylamines; Mice; Monensin; Muramidase; Peptides; Protein Binding; T-Lymphocytes; Temperature

The participation of lysosomal enzymes, hydroxyl radicals, and mitochondrial respiration in the cytocidal effect of TNF on tumor cells was investigated. The cytotoxicity of TNF on L-M cells was clearly reduced by lysosomotropic agents, DMSO (hydroxyl radical scavenger), NDGA (lipoxygenase inhibitor), and sodium azide (mitochondrial respiration inhibitor). The results suggest that lysosomal enzyme and hydroxyl radicals play an important triggering role in the destruction of tumor cells by TNF, and that the process of destruction might require ATP.

Topics: Ammonium Chloride; Animals; Chloroquine; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Free Radicals; Hydroxides; Hydroxyl Radical; Leupeptins; Masoprocol; Methylamines; Mice; Muramidase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha