Slow Transients in the Activity of the NAD Malic Enzyme from Crassula

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Abstract
The NAD malic enzyme from C. argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or .gamma. ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoe daigremontiana has a similar lag. The lag is greatest in freshly prepared enzyme and diminishes with storage at -70.degree. C, but the activity of the enzyme also diminishes with storage. The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached. Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO42- in the assay substantially decrease the lag. The lag is shorter with Mn2+ as the required divalent cation than when Mg2+ is used. The response of enzyme activity to pH shows that the intrinsic activity is greater with Mg than with Mn, although the rate actually attained is lower with Mg2+ because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn2+ is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH valoes. Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme.