Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.