THE PROTEIN-KINASE, PROTEIN PHOSPHATASE AND INHIBITOR PROTEIN OF NITRATE REDUCTASE ARE UBIQUITOUS IN HIGHER-PLANTS AND INDEPENDENT OF NITRATE REDUCTASE EXPRESSION AND TURNOVER

Nitrate reductase activity and NR protein levels in various leaf tissu es were drastically decreased (< 3.5% of normal activity) either by ke eping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the s ole N-source, or by germinating and growing etiolated seedlings in com plete darkness (squash). The presence of nitrate reductase protein kin ase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalte d extracts to inactivate (ATP-dependent) and reactivate (5'-AMP/EDTA-d ependent) added purified spinach NR in vitro. Extracts from low-NR pla nts (ammonium-grown pea and squash) were also prepared from leaves har vested at the end of a normal light or dark phase, or after treating l eaves with anaerobiosis, uncouplers or mannose, conditions which usual ly activate NR in nitrate-grown normal plants. Without exception, extr acts from NR-deficient plant tissues were able to inactivate and react ivate purified spinach NR with normal velocity, irrespective of pretre atment or time of harvest. Considerable NRPK, NRPP and IP activities w ere also found in extracts from almost NR-free ripe fruits (cucumber a nd tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partiall y purified with normal yields from NR-deficient squash or spinach leav es, following the purification protocol worked out for nitrate-grown s pinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squa sh or spinach; phosphorylated added purified spinach NR with gamma-[P- 32]ATP and inactivated the enzyme after addition of IP. It is suggeste d (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR a re rather species- or organ-unspecific, (ii) that they do not turn ove r as rapidly as does NR, (iii) that they are probably expressed indepe ndently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed b y desalting.