THE EFFECT OF CALCIUM CHELATORS ON MICROSOMAL PYRIDINE NUCLEOTIDE-LINKED DEHYDROGENASES OF SUGAR-BEET CELLS

Cell suspension cultures of Beta vulgaris L., treated with calcium chelators or untreated, were used to characterize pyridine nucleotide-dependent diaphorases of microsomes. The microsomal activity of NADH-dependent duroquinone reductase from cultures treated with 10 mM Na(2)EGTA for 24 h increased by a factor of 1.8 with respect to control microsomes, and was mainly associated with particles of d=1.17 g ml(-1). NADPH-duroquinone reductase and NADH-ferricyanide reductase activities showed smaller increases. Bacterial protein-lipopolysaccharide complexes (prLPS) also promoted the increase of microsomal diaphorases; CaEGTA was ineffective. EGTA effects on enzymes of supernatant and mitochondria were negligible, although Na(2)EGTA treatment induced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with 1% LPC and fractionated in high-efficiency gel permeation columns (FPLC) the diaphorase activities were found associated to three major proteins: (i) NADH-specific quinone reductase (NADH-QR) of 340 kDa; (ii) pyridine nucleotide-nonspecific quinone reductase (NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity; (iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38 kDa. The microsomes from EGTA-treated cells also showed a highly active NADH-QR having a larger molecular mass (440 kDa) than in control cells. NAD(P)H-QR also showed increased activity. We conclude that external Ca2+ chelation induces changes in dehydrogenase components in microsomes. Furthermore, prLPS probably exert part of their effect on plants through Ca2+ chelation.