The subcellular distribution of rat liver serine-pyruvate aminotransferase.

1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a 'total-particles' fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.     

The subcellular distribution of fumarase isozymes in rat liver

Between 13 and 25% of the fumarase activity of rat liver was found to be cytosolic in origin the remainder being localised in the mitochondria. Electrophoretic analysis on cellulose acetate showed that mitochondria do not contain detectable levels of cytosolic isozyme or vice versa.     

Uptake and subcellular distribution of injected transferrin in rat liver

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Thymidine kinase of rat liver. Activation by phospholipase C and subcellular distribution in the liver

1. The thymidine kinase activity of rat liver is greatly enhanced on addition of phospholipase C to the assay mixture. 2. Most of the thymidine kinase activity of the liver is recovered in the mitochondrial and in the impure ;nuclear' fractions. No activity was detected in purified nuclei prepared in high-density sucrose. 3. A substantial thymidine kinase activity could be detected, with the aid of phospholipase C, in all rat tissues examined.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

Levels and subcellular distribution of endogenous corticosterone in rat liver during development

The concentration of corticosterone in liver homogenates, liver cytosol and purified nuclear fractions, and in plasma of fetal, newborn, immature and adult rats has been measured by radioimmunoassay.Highest plasma corticosterone levels were found in fetal rats, decreasing close to the levels observed in the adrenalectomized rat by the 6th day of postnatal life followed by a rise in the adult rat. The concentration of corticosterone in liver during development paralleled the plasma levels, the liver to plasma corticosterone ratio ranging between 0.09 and 0.17 suggesting that the corticosterone retained by the tissue is related to the unbound fraction of the hormone in plasma.Both plasma and tissue corticosterone levels declined after adrenalectomy and they were elevated after ether stress.Fractionation of liver homogenates showed that the major fraction of liver corticosterone is localized in the cytosol. Purified liver nuclei contained between 9 and 16% of the total liver corticosterone. The amount of corticosterone in the nuclei seems to be related to the plasma and tissue hormone levels rather than the concentration of cytoplasmic glucocorticoid receptors. Since most of the nuclear corticosterone appears to be bound to receptors, it has been calculated that close to 60% of the cellular receptors in fetal liver are localized in the nucleus. In adult rat liver, only about 10% of the cellular receptors appear to be associated with nuclei. Changes in the concentration of glucocorticoid receptors in liver during development and after adrenalectomy are inversely related to changes in plasma corticosterone levels. It is suggested that corticosterone may regulate the levels of its own receptors in liver.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Effect of gold thioglucose on subcellular selenium distribution in rat liver and kidney

Biological Trace Element Research - Reported are the subcellular distributions of selenium (Se), gold, glutathione peroxidase, and enzyme markers for nuclei, mitochondria, lysosomes, and soluble...     

The development of lysosomal apparatus. II. Incorporation,subcellular distribution,and intraparticulate hydrolysis of 131 I-albumin by liver of mice at perinatal stages

Mouse fetuses at 15th and 18th day of gestation, and newborns aging 0, 5, 10, and 15 days were injected i.p. with 131 I-albumin (RISA). The degree of incorporation by liver and the intraparticulate hydrolysis of RISA in 27,000g × 10 min. particles increased after birth. By this time, changes in subcellular distribution of RISA and marker enzymes were also observed. Following an i.p. injection of India ink, numerous hepatic cells stained with carbon particles were observed at the light microscope from day 5 of life. The results suggest that the lysosomal apparatus acquires full capacity to incorporate colloidal particles and to degrade foreign macromolecules in the first week of life.     

The effect of thyroidectomy on the subcellular Mg distribution in rat liver

The subcellular distribution of Mg²⁺ in livers of normal and thyroparathyroidectomized rats has been studied. Significant increases in Mg²⁺ are found in the mitochondrial fractions of thyroparathyroidectomized rats accompanied by a decrease in the Ca²⁺ content. In the microsomal fractions no significant modifications of both ions were detected. Propylthiouracil administration reproduced the ionic alterations found after surgical thyroidectomy and a triiodothyronine replacement therapy to thyroidectomized rats resulted in a reversion of the Mg²⁺ content back to normal levels. The possible participation of the parathyroid in the above mentioned phenomena could be excluded in the present work.     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.