Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

Postnatal changes of some enzymatic activities of energy supplying metabolism in the cortex, inner and outer medulla of the rat kidney

1. In rat kidney cortex, outer and inner medulla the development of activities of seven enzymes was investigated during postnatal ontogeny (10, 20, 30, 60 and 90 days of age). The enzymes were selected in such a manner, as to characterize most of the main metabolic pathways of energy supplying metabolism: hexokinase (glucose phosphorylation, HK), glycerol-3-phosphate dehydrogenase (glycerolphosphate metabolism or shunt, GPDH), triose phosphate dehydrogenase (glycolytic carbohydrate breakdown, TPDH), lactate dehydrogenase (lactate metabolism, LDH), citrate synthase (tricarboxylic acid cycle, aerobic metabolism, CS), malate NAD dehydrogenase (tricarboxylic acid cycle, intra-extra mitochondrial hydrogen transport, MDH) and 3-hydroxyacyl-CoA-dehydrogenase (fatty acid catabolism, HOADH). 2. The renal cortex already differs metabolically from the medullar structures on the 10th day of life. It displays a high activity of aerobic breakdown of both fatty acids and carbohydrates. Its metabolic capacity further increases up to the 30th day of life. 3. The outer medullar structure is not grossly different from the inner medulla on the 10th day of life. Further it differentiates into a highly aerobic tissue mainly able to utilize carbohydrates. It can, however, to some extent, also utilize fatty acids aerobically and produce lactate from carbohydrates anaerobically. 4. The inner medullar structure is best equipped to utilize carbohydrates by anaerobic glycolysis, forming lactate. This feature is already pronounced on the 10th day of life, its capacity increases to some extent during postnatal development, being highest between the 10th and the 60th day of life.     

Localization of insulin degradation products to an intracellular site in isolated rat hepatocytes

In the present study, we have examined whether insulin degradation products are present on the surface of isolated rat hepatocytes and can be removed by an acid dissociation technique. Hepatocytes were incubated with [125I]insulin for 30 minutes, rapidly washed to remove unbound insulin, and then briefly exposed to acidic conditions (pH 5.0) to remove bound hormone from the cell surface. The radioactive material removed from the cell by acid dissociation and that remaining with the cells were separately analyzed by high performance liquid chromatography. The two primary degradation products of insulin present in control cell extracts were found only with the cell-associated material after acid dissociation. The insulin-sized radioactive material in the extract of acid-dissociable material consisted of only intact [125I]insulin. These results show that the two primary degradation products of insulin in rat hepatocytes are found only intracellularly and suggest that the degradation of the hormone begins after it is internalized.     

Selective loss of uncoupling protein mRNA in brown adipose tissue on deacclimation of cold-acclimated mice

The time course of changes in the level of uncoupling protein mRNA when cold-acclimated mice were returned to a thermoneutral environment (33 degrees C) was examined using a cDNA probe. Upon deacclimation, there was a marked loss of uncoupling protein mRNA within 24 h, which precedes the loss of uncoupling protein from mitochondria. This loss of uncoupling protein mRNA was selective, since there was no change in the relative proportion of cytochrome c oxidase subunit IV mRNA or poly(A)+ RNA in total RNA. The results suggest that the decrease in the mitochondrial content of uncoupling protein during deacclimation is likely the result of turnover of existing protein, with very little replacement due to a lower level of its mRNA.     

Do myc, fos and E1A function as protein phosphatase inhibitors?

The oncogenic proteins myc, fos and E1A bear striking resemblance to protein phosphatase inhibitors 1 and 2. Both sets of proteins possess several regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). In addition to PEST sequences four of the five proteins contain clusters of arginine-arginine pairs. On the basis of these similarities, I suggest that myc, fos and E1A are protein phosphatase inhibitors.     

Changes in the size of the myocardial damaged area in postischemic reperfusion

The changes in the size of the myocardial injury area during reperfusion after the coronary occlusion-induced ischemia lasting 30 minutes are phasic in nature. Until 3.5 h the injured area increases and after 23.5 h relatively diminishes. After a more prolonged ischemia such manifestations are either unmarked or absent. Ischemia lasting from 30 min to 4 hours followed by reperfusion, as compared with ischemia of the same duration without reperfusion, normally gives rise to the formation of an area of injury, which is less or occasionally equal in size. The data obtained and reported indicate that in the area of coronary occlusion there are groups of cardiomyocytes that differ as regards the resistance to ischemia.     

Induction of electrogenic phosphate transport through the mitochondrial membrane by a thermostable cytoplasmic factor in hyperthyroidism

Addition of a thermostable cytoplasmic fraction leads to the uncoupling of oxidative phosphorylation of the mitochondria. In hyperthyrosis such an effect manifests itself more powerfully than in the control. Addition of the thermostable cytoplasmic fraction induces electrogenic phosphate transport via the mitochondrial membrane. In hyperthyrosis, the activity of the thermostable inducer of phosphate transport in the cytoplasm increases. The functioning of the phosphate cycle may be the cause of the uncoupling of oxidative phosphorylation of the mitochondria during the disease in question.