Inner- and outer-membrane enzymes of mitochondria during liver regeneration

1. Marker enzymes for the mitochondrial matrix, inner membrane, inter-membrane space and outer membrane were measured in mitochondria isolated from control and regenerating rat liver. The specific activity of these enzymes was then followed for up to 30 days after operation. 2. The specific activity of marker enzymes for the matrix, inner membrane and inter-membrane space remained constant during liver regeneration. 3. However, the specific activities of monoamine oxidase and kynurenine hydroxylase, both outer-membrane markers, fell by 67% and 49% respectively from their control values at 4 days after operation, and returned to normal by about 3 weeks. 4. The repression of kynurenine hydroxylase activity was shown to be unrelated to any independent variation in tryptophan catabolism, based on tryptophan pyrrolase assays. 5. These results are considered to indicate that enzymes of the inner and outer mitochondrial membranes are synthesized asynchronously during morphogenesis. 6. The enzyme complement of purified outer membrane at 4 days after operation was about 50% of that of the appropriate control. Thus the composition of the outer membrane itself may vary dramatically, and supports the concept that constitutive enzymes may turn over independently of a membrane's existence. 7. The behaviour of the rotenone-insensitive, NADH cytochrome c reductase did not parallel the other outer-membrane enzymes for intact mitochondria, but did so when assayed in highly purified fractions of outer membrane. This suggests a labile binding to the outer membrane during the early stages of morphogenesis. 8. Electrophoresis of inner- and outer-membrane proteins revealed little difference between control and experimental mitochondria at 4 days, except for an increase in several, high-molecular-weight components of the outer membrane. These bands closely correspond to similar bands derived from smooth endoplasmic reticulum. 9. The results are discussed in relation to the biogenesis and turnover of mitochondria, and are considered to provide evidence for turnover as a unit, at least for the matrix, inner membrane, inter-membrane space and possibly some form of primary outer membrane.     

Effect of thyroid status on microsomal enzymes during liver regeneration in the rat

The effects of thyroid status upon cyt. P 450 concentration and ethoxycoumarin deethylase, benzopyrene hydroxylase and UDP-glucuronosyltransferase activities in the liver microsome fraction were far more important in partially hepatectomized rats than in control animals. The partial hepatectomy simultaneously lowered the MFO enzymes activities in the hepatic microsome fraction and made them more sensitive to thyroid hormones effects.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Histone phosphorylation during liver regeneration.

The pattern of histone phosphorylation at acid-stable, alkali-labile sites has been examined throughout the early stages of liver regeneration, namely at times of “gene activation”. Among the histones, only H1 shows an increase in phosphorylation. This increase initiates near the end of the period of chromatin template activation. Thus, there is no obvious temporal correlation between increased histone phosphorylation and increased RNA synthesis. The relative levels of phosphorylation of the various histones and the change in H1 phosphorylation observed in the liver system closely parallel the patterns exhibited by cultured animal cells during the G1 and S phases of the cycle as described by other investigators.     

Phospholipid metabolism during regeneration of rat liver

The concentration and composition of phospholipids and mitotic activity in regenerating rat liver were studied. (1) The total amount of liver phospholipid increased approximately linearly during 48h after operation but without change in the relative concentrations of individual phospholipids. (2) The appearance of mitoses 30h after operation was accompanied by an increased incorporation of (32)P into the liver phospholipids. (3) The regenerating livers incorporated a higher percentage of the label into the phosphatidylserine+phosphatidylinositol fraction than those of control rats. The percentage of the label incorporated into phosphatidylethanolamine in these livers increased but decreased in the phosphatidylcholine.     

Membrane potential measurements during rat liver regeneration

Membrane potential was measured in perfused rat liver and was shown to increase from ?33 ± 1.0 mV in livers from normal rats to ?50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.     

Changes in lysosomal cathepsins during liver regeneration

The effect if $\text{2}\text{3}$ hepatectomy in the adult rat on the specific and total activities of two lysosomal endopeptidases, cathepsins B₁ and D, has been examined. The specific activity of both enzymes fell rapidly following hepatectomy when compared to paired, sham-operated controls. When changes in total protein were compared to changes in cathepsins B₁ and D, all three decreased in a parallel fashion for the first 18 h. At 24 h, total liver protein increased rapidly while cathepsins B₁ and D continued to decrease. Cathepsin B₁ fell to a level of 12% of non-operated control levels at 36 h, while total protein was already back to 40% of control levels. In contrast to the decreases in the activities of the cathepsins, there was an increase in the activities of lactate dehydrogenase and malate dehydrogenase during the first two days of regeneration. The clear lag in replacement of the cathepsins relative to other liver proteins following partial hepatectomy suggests that cathepsin activity is selectively controlled and that lowering the levels of cathepsins B₁ and D may play an important role in the decreased degradation of protein seen during the early phases of liver regeneration.