1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.

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SUMMARY : Treatment of the cells of various Bacillus spp. with an alkaline solutbn of sodium hypochlorite resulted in dissolution of the cells and liberation of the intracellular lipid inclusion bodies. Analyses of the isolated and purified inclusions of Bacillus cereus grown under a variety of cultural conditions showed them to contain about 89 yo poly-P-hydroxybutyrate and 11 yo ether-soluble lipid. Parallel estimations of the poly-P-hydroxybutyrate content of intact organisms by Lemoigne's chloroform extraction method showed that all of it was present in the lipid inclusions.These observations form the basis of a simple and rapid method of estimating the poly-/3-hydroxybutyrate content of Bacillus spp. It consists essentially of digesting a washed bacterial suspension with a standard alkaline hypochlorite solution under standard conditions and measuring the residual turbidity.The intracellular lipid inclusions which form such a prominent feature of the large-celled species of BaciEZw have been little studied. Their avidity for 03-soluble dyes such as Sudan Black clearly indicates their content of 'fatty' substances, but little is known of the nature of these or other materials that may be present. A notable advance was made by Lemdgne, Delaporte & Croson (1954) who were able to correlate the occurrence of lipid inclusians in BaciZZw spp. with the presence in the cells of appreciable amounts of a polymer of /3-hydroxybutyric acid, previously isolated from Bacillus ' M ' by Lemoigne (1927). On the basis of these findings the authors concluded that the polymer was a constituent of the inclusions. Their view received some support from the observations of Weibull(1953), but has yet to be directly confirmed, The purpose of this paper is to provide such confirmation, and at the same time to describe a rapid method of estimating the poly-P-hydroxybutryate content of small samples of organisms. The method is based on the observation of Meyer (1901) that when organisms of Bacillus spp. are suspended in an alkaline solution of sodium hypochlorite, they are almost completely dissolved, only the lipid inclusions remaining intact. METHODSOrganisms. The laboratory strain of BacdZzls cereus (strain SC) used had the following properties : motile ; Voges-Proskauer positive ; catalase positive ; rapid liquefaction of gelatin and coagulated serum ; ,8-haemolytic ; produced acid on glucose, sucrose, maltose, glycerol and salicin but not on lactose, dukitol, mannitol, arabinose, rhamnose, xylose, raffinose or inulin. Abundant

1. The activities of hydroxymethylglutaryl-CoA synthase and lyase in rat liver were found to be two- to 15-fold greater than those reported by other authors under similar conditions. 2. When expressed on the basis of body weight, no appreciable differences were found between the activities of hydroxymethylglutaryl-CoA synthase in whole homogenates of livers from normal and starved rats. The synthase activity increased by 70% and 140% in livers of alloxan-diabetic rats and rats fed on a high-fat diet respectively. 3. Hydroxymethylglutaryl-CoA lyase activity showed no significant increases in starvation or alloxan-diabetes, but a 40% increase was found in fat-fed rats. 4. Less than 12% of the activities of both enzymes were found in the cytoplasmic fraction of normal liver. The cytoplasmic activity doubled in alloxan-diabetes and starvation; on feeding with a high-fat diet the increase, though significant, was less marked. 6. The intracellular distribution of glutamate dehydrogenase indicated that the changes in the cytoplasmic activities observed were not due to leakage from the mitochondria. 7. Feeding with a normal or high-fat diet after 48hr. starvation caused within 24hr. a decrease in the cytoplasmic activity of hydroxymethylglutaryl-CoA synthase to values lower than those found in rats fed on a corresponding diet for a longer period of time. 8. Acetoacetyl-CoA deacylase activity in liver was about 20% of that of hydroxymethylglutaryl-CoA synthase and was primarily located in the cytoplasm. Starvation or alloxan-diabetes did not alter the acetoacetyl-CoA deacylase activity. 9. It is concluded that variations in the concentrations of enzymes involved in acetoacetate synthesis play no major role in the regulation of ketone-body formation in starvation and alloxan-diabetes. The changes in the cytoplasmic activities of hydroxymethylglutaryl-CoA synthase and lyase suggest that acetoacetate synthesis can occur in the cytoplasm. This may play a role in the disposal of surplus acetyl-CoA arising in the cytoplasm when lipogenesis is inhibited.