DNase activity of micrococcal endonuclease covalently immobilized on nylon and polystyrene.

Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes. The activity, evaluated by hydrolysis of high-molecular-weight and supercoiled DNA, indicates that both derivatives are highly stable for storage and further use. Immobilization of the enzyme is much more effective when the covalent binding is performed on polystyrene. By using different activation methods with these matrices, a set of immobilized nucleases with various levels of enzymatic activity can be prepared. The possibility of working in a wide range of enzymatic activity and at low temperature and Ca2+ concentrations in different buffers makes these immobilized nucleases very useful for investigating accessible DNA regions in chromatin structure.

Enzyme coupling method on calibrated nylon spheres: application to the selective trypsinization of histones in chromatin.

A new method consisting of a two-step activation was developed in order to covalently immobilize enzymes on calibrate nylon 66 spheres. This efficient method associates for the first time peptide bond cleavage and O-alkylation of the support. Optimal conditions for activation and protein coupling were defined, and immobilized trypsin was used to investigate the histone accessibility on chromatin. This approach, which allows us to degrade first progressively H1, indicates that H4 seems inaccessible both in relaxed and condensed chromatin.

Conductimetry: a new tool for studying inhibition of elastolysis.

The conductimetric method was applied to the measurement of human leukocyte elastase activity, using insoluble elastin as a substrate. From conductance changes, initial rates of elastolysis were derived. A linear relationship of enzyme activity with enzyme concentration was demonstrated up to 400nM of enzyme, for three different substrates. In this concentration range, inhibition of elastolysis by eglin c was studied for different concentrations of eglin c. A 50% inhibitory concentrations of 0.13-0.15 microM of eglin c was derived from our results, corresponding to an inhibitor/enzyme ratio of about 0.5, indicating a strong inhibition, as previously demonstrated by authors using synthetic substrates.

Ca2+ and Mg2+ protection against thermal denaturation of pancreatic elastase.

Thermal denaturation of porcine pancreatic elastase was studied by difference spectrophotometry. At 293 nm, and pH 8.0, the thermal transition of elastase occurs with a midpoint temperature (Tm) of (58.0 +/- 0.5) degrees C. Mg2+ and Ca2+ stabilize the native form in increasing the midpoint temperature of the transition, Ca2+ being more effective than Mg2+ in the 0-0.02 M concentration range. Furthermore, Ca2+ protects pancreatic elastase against the destabilizing effect of Cu2+. Whatever be the temperature between 40 degrees C and 55 degrees C, Ca2+ protects pancreatic elastase against loss of enzymatic activity.

Lipase assay in duodenal juice using a conductimetric method.

Lipase activity in duodenal juice is known to undergo important variations in pathologic states, especially in cases of chronic pancreatitis. Almost all of the current assay methods are based on the measurement of hydrolysis of olive oil or triolein, mainly by potentiometry. As we have developed a conductimetric method for enzyme activity measurements, we have applied it to lipase assay. A higher experimental conductimetric sensitivity is obtained when liberated acids have a short chain (higher limiting equivalent conductivity). We have therefore used triacetin as a substrate and compared out method with potentiometry (pH-stat) and spectrophotometry. The correlation coefficients of both methods with conductimetry were 0.94 and 0.97, respectively, indicating that the conductimetric method may be used for lipase assay in duodenal juice, using triacetin as a substrate.

[Fixation of antibodies by covalent bonds to polystyrene: use of the immunoenzyme technic for the detection of Escherichia coli K99 in calf feces]. / Fixation d'anticorps par liaisons covalentes sur le polystyrène: application de la technique immunoenzymatique à la recherche de Escherichia coli k99+ dans les fèces de veaux.

A solid phase enzyme immunoassay using antibodies covalently bound to polystyrene balls is developed. Results show the same sensitivity as the method with antibodies adsorbed to solid support. By means of K99+ E. coli detection on diarrhoeic calves, results are compared with bacteriological findings. The interest of the method is discussed.