Oxidized and reduced nicotinamide-adenine dinucleotide phosphate in tissue suspensions of rat liver.

Abstract

1. The concentrations of NADP and NADPH(2) in homogenates of rat liver (expressed as mug./g. wet wt. of tissue homogenized) were compared with values obtained from intact samples of liver taken from the same female rat. With 0.25m-sucrose alone as the suspending medium, or in combination with tris buffer or 0.01-0.1m-nicotinamide, considerable decreases in the sum of the NADP+NADPH(2) concentrations were occasionally observed during 30min. storage of homogenates at 0 degrees . However, addition of 0.5m-nicotinamide+5mm-tris buffer to 0.25m-sucrose for use as a suspending medium maintained the sum of the NADP+NADPH(2) concentrations in homogenates at the level found in intact tissue for at least 30min. at 0 degrees . 2. The effects of freezing intact tissue and homogenates in liquid nitrogen before the extraction of NADP and NADPH(2) were studied. Freezing alone appears to convert a significant amount (approx. 30%) of liver NADPH(2) into an equivalent amount of NADP in intact tissue. This is discussed in terms of the ;bound NADP' reported by Burch, Lowry & Von Dippe (1963). 3. The intracellular distributions of NADP and NADPH(2) in intracellular fractions of rat liver were studied by using a modified centrifuging scheme that allows extraction of the isolated fractions to be performed within 45min. of killing the animal. Approx. 50% of the total NADP+NADPH(2) was found in the large-particle fractions and the remaining 50% was mostly in the soluble fraction of the cell. 4. Further investigations are reported on the nature of ;bound NADP' in rat liver. Most of this material appears associated with the ;nuclear' (containing nuclei, debris, erythrocytes etc.) or large-mitochondrial fractions, or both, obtained by low-speed centrifuging of rat-liver homogenates. 5. Although in some experiments the variations produced in the concentration of NADPH(2) present in large-particle fractions were followed by similar changes in that of ;bound NADP', in other cases no such direct relationship was obtained. Addition of phenazine methosulphate, for example, consistently lowered the concentration of NADPH(2) yet raised the concentration of ;bound NADP' in rat-liver mitochondrial fractions.