Partial purification of two proteins (100 kDa and 67 kDa) cooperating in the ATP-dependent inactivation of spinach leaf nitrate reductase

Using a three-step purification procedure, two protein fractions which catalyzed the ATP-dependent in-activation of nitrate reductase (NR) were obtained from spinach (Spinacia oleracea L.) leaf extracts. Purification involved ammonium-sulfate fractionation, anion-exchange chromatography and size-exclusion chromatography. The capacity of the fractions to inactivate NR by preincubation with ATP was examined by using as target either a crude NR-ammonium sulfate precipitate or partially purified NR (ppNR). The fractions were also examined for protein-kinase activity by measuring the phosphorylation of histone III S (or casein) with-[³²P]ATP as substrate, and subsequent SDS-PAGE, autoradiography and liquid scintillation counting of cut-off histone bands. The two proteins had apparent molecular weights in the 67-kDa and 100-kDa region (termed P₆₇ and P₁₀₀, respectively). Neither P₆₇ nor P₁₀₀ alone was able to inactivate ppNR by preincubation with ATP. However, when P₁₀₀ and P₆₇ were added together to ppNR, ATP-dependent inactivation was observed, with a half-time of about 10 min. The P₆₇, but not P₁₀₀ had histone-kinase activity (casein was not phosphorylated). Using the partially purified system, various compounds were examined as possible effectors of NR inactivation. Sugar phosphates had little effect on the inactivation of NR. Addition of AMP at very high concentrations (5 mM), and removal of Mg²⁺ by excess EDTA also prevented the inactivation.Abbreviations AS ammonium sulfate - DTT dithiothreitol - NR NADH-nitrate reductase - NRA nitrate reductase activity - ppNR partially purified nitrate reductase