We examined the effects of temperature on the activity and steady-state kinetic properties of alkaline phosphatase (EC 3.1.3.1). Purified isoenzymes from human liver, intestine, and placenta were used, as was human serum, and the enzyme from porcine kidney. Phosphatase activity was estimated by two different assay techniques. For all isoenzymes, apparent Michaelis constants for the substrate 4-nitrophenyl phosphate decreased with increased temperature; Km at 37 degrees C was typically half that determined at 25 degrees C. All enzymes of human origin exhibited similar linear Arrhenius relationships over the range examined, 20-37 degrees C (Ea of 30-36 kJ X mol-1). The porcine kidney enzyme obeyed an Arrhenius relationship that was slightly, but significantly, different from the isoenzymes of human origin. Temperature relationships based upon Arrhenius behavior and individual activity measurements are presented. For human alkaline phosphatases, they differed by no more than 10%.
An unusual nonenzymic prothrombin activation was firmly established by demonstrating thrombin formation in mixtures of synthetic ply-L-lysine (plyLYS) and homogeneous horse prothrombin ( 1 ) . Reaction characteristics were a high, optimum weight ratio of synthetic protein to prolthrombin, a low temperature optimum, high sensitivity to ionic strength changes, and insensitivity to diisupropylphosphofluoridate (DFP) . Several basic materials now substitute for the polyLYS, including synthetic ply-L-ornithine (polyORN) and polyrnyxin B. The prothrombin activation in protamine sulfate solution, for which an autmtalytic mechanism was suggested (2), has several characteristics of this form of nonenzymic reaction.Nonenzymic prothrombin activation is inhibited by heparin, poly-L-glutadc acid ( polyGLU) , and ply-L-aspartic acid ( p l y -ASP).Materials and methods. Horse prothrombin isolated from citrated horse plasma in 60% yield was thrombin-free and immunochemically and physicochemically homogeneous after equilibrium ion-exchange chromatography (3,4).
Clinical laboratories estimating enzyme activity in serum are using commercial lyophilized sera for four major purposes. These uses—as a standard and for intermethod, intramethod, and precision control—are segregated, and specifications for each deployment are examined in terms of requirements for the enzyme material: freedom from interfering or indicator enzymes and chromogens; high specific activity; inclusion of optimal cofactor concentrations; commutability, human properties and source; the presence of a single isoenzyme; and stability. The effects of serum matrix and variable assay conditions on the utility of enzyme materials are analyzed. Specifications differ for each enzyme material application. The compatibility of commercial lyophilized sera containing aspartate aminotransferase activity with several cited specifications is assessed
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