Increase In Free Activity Research Articles

Cationic lipids destabilize lysosomal membrane in vitro

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)- N, N, N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of β-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Enzymatic activities in the iris-ciliary body of the rabbit eye during experimentally induced acute ocular inflammation.

Intravitreal injection of 5 micrograms of Shigella endotoxin, in the rabbit eye, induced an acute inflammatory response which was characterised by conjunctival hyperaemia, limbal and ciliary vascular injection, iritis, aqueous flare, miosis and reduction in intraocular pressure. Iris-ciliary body tissues, from normal and inflamed eyes, were fractionated into subcellular enriched fractions and the activities and distribution of selected enzymes were estimated. Alkaline phosphatase, a plasma membrane-bound enzyme, showed an increase in activity, whereas succinate dehydrogenase and Mn-Superoxide dismutase, both mitochondrial-bound enzymes, exhibited decreased activities. Lysosomal acid phosphatase displayed an increase in free activity and retention of latent activity inside the organelle. No alteration in free activity was shown by acid cathepsin. The cholinesterases did not exhibit any changes in activities nor did the cytosolic enzymes Cu/Zn-superoxide dismutase and lactate dehydrogenase. The decrease activity of the respiratory mitochondrial enzyme succinate dehydrogenase may contribute to the reduction in intraocular pressure, and the ability of the lysosomal organelles to retain their hydrolytic enzymes, ensures recovery of the cell from acute inflammatory attack.

Effect of a Protein-Free Diet and Fasting on RNA Polymerase Activity in Mice

The objective of this study was to measure the effect of diet on the distribution of RNA polymerase activity in mice kidney. Bound RNA polymerase activity was decreased in kidneys of fasted mice. During a short fast (16 hours), this decrease in bound RNA polymerase I activity was accompanied by an increase in free activity and suggested a possible interference in binding as the reason for decreased bound activity. Bound RNA polymerase II also decreased but with no increase in free activity. During longer fast times (40 hours), the bound RNA polymerase activity (RNA polymerase I and II) remain depressed and the free activity returns to control levels. Fasting decreased bound RNA polymerase I in liver by 41% after 40 hours but had little effect on the free activity. Liver of mice, fed a protein-free diet, were found to have an increased RNA polymerase activity due to a large increase in free RNA polymerase I activity. All other liver RNA polymerase activities were unchanged. In contrast, kidneys of mice fed a protein-free diet, have decreased RNA polymerase activity due to a decrease both in bound RNA polymerase I and II; free RNA polymerase is unchanged. These results demonstrate that diet has a very selective effect on RNA polymerase activity.RNA polymerase protein-free diet free and bound RNA polymerase mouse liver kidney nuclei

Modification of rat liver fructose biphosphate aldolase by lysosomal proteinases

In vivo proteolytic modification of liver aldolase on administraton of leupeptin, a thiol proteinase inhibitor of microbial origin, is reported. When leupeptin was injected into rats, the activity of aldolase in the liver decreased to 40% of that in control rats. Molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that a decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. Injection of leupeptin also caused marked increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D and moderate increase of cathepsin B and cathepsin L. Increase in free activity of cathepsin A returned to the level of control rats by 12 hr after injection of leupeptin, whereas 36 hr was required for recovery of decreased aldolase activity. When insulin was coinjected with leupeptin, increase in the activity of free cathepsin A and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to action of a lysosomal protease(s). Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin. The aldolase inactivating proteinase in the lysosomal fraction was inhibited by PMSF and leuptpin and not by pepstatin. Purified cathepsin A (a serine proteinase), cathepsin B and cathepsin L (thiol proteinase) are potent inactivators of aldolase but cathepsin H and cathepsin D are not. Cathepsin A, B and L are involved in activation of aldolase in lysosomes. Endogenous thiol proteinase inhibitor which inhibits lysosomal thiol proteinases (cathepsin B, L and H) is found in the cytosol fraction of liver. The level of thiol proteinase inhibitor actually decreased to 60% of that in control rats in leupeptin-treated rats, suggesting that non-thiol proteinase cathepsin A is a major factor in inactivation of aldolase in lysosomes. Not only leupeptin but also other proteinase inhibitors (antipain, E-64-D, chloroquine) caused increase of labilization of the lysosomes and decrease in aldolase activity. Physiological stimuli which are known to induce the labilization of the lysosomal membrane, such as starvation and glucagon, caused slight or no significant increase of activities of free cathepsin A and D and resulted in no apparent change in aldolase activity.

Nutrition and lysosomal activity. The influence of the vitamin A status on the proteolytic activity of extracts from the livers and kidneys of rats.

1. Young rats were kept for several weeks on a diet deficient in vitamin A. Some were undosed, others were given marginal (25i.u. weekly), adequate (1000i.u. weekly) or excessive (50000i.u. daily) doses of vitamin A acetate. The undosed rats developed signs of vitamin A deficiency, and the overdosed animals had skeletal fractures indicative of hypervitaminosis A. 2. The rats were decapitated. Their livers, and sometimes their kidneys, were homogenized and processed by centrifugal methods to sediment most of the lysosome fractions. Proteolytic activity was measured, after treatment with a detergent, in the whole homogenate (;total' activity), in the pellet obtained after 20min. at 15000g (;bound' activity) and, without treatment with detergent, in the supernatant (;free' activity). 3. In rats suffering from hypervitaminosis A the free activity and to a smaller extent the total activity were increased. Free activity was also raised in most rats suffering from avitaminosis A, but less than in those suffering from hypervitaminosis. 4. The vitamin A status appeared to have little effect on the proteolytic activity of the kidneys. Results for total and free activities, but not for bound activities, were higher than for corresponding liver preparations. 5. Control experiments were done on starved rats and on rats which were pair-fed with hypervitaminotic animals. Short periods of starvation caused an increase in free activity in young rats, but not in adults. The increases caused by starvation were much less than those caused by hypervitaminosis A. 6. For studies of the distribution of vitamin A more complete separation of the subcellular fractions was carried out on the combined livers from several hypervitaminotic rats. The concentration of vitamin A in the lysosome fraction was less than in the liver as a whole. 7. Our finding that the free proteolytic activity of the liver is increased by toxic oral dosing with vitamin A can be considered an extension of the previous observation that proteolytic enzymes are liberated when lysosomes are treated in vitro with vitamin A.

A study of some acid hydrolases in the liver of the larval and adult lamprey.

The authors studied the behaviour of two acid hydrolases (phosphatase and proteinase) in the liver of the larval form (ammocoetes), of the starved larva, and of the adult lamprey (Lampetra zanandreai, Vladykov). In the homogenates of liver of ammocoetes in 0.25 M sucrose the enzymatic activities are largely sedimentable (phosphatase 67%; proteinase 56%). In the starved ammocoetes and in the adult lamprey the percentage of sedimentable activity gradually falls (phosphatase 46% and proteinase 52% in the starved ammocoetes; phosphatase 44% and proteinase 49% in the adult lamprey) whilst there is a corresponding gradual increase in free activity (phosphatase: from 24% in the normal ammocoetes to 39% in the starved ammocoetes and 50% in the adult lamprey; proteinase: from 25% in the normal ammocoetes to 35% in the starved ammocoetes and 54% in the adult lamprey). The action of detergent Triton X-100 causes an equal distribution of hydrolases activity in the three conditions of the liver. Only 25% of the sedimentable acid glycerophosphatase is accessible to the substrate in the ammocoetes, whilst in the starved ammocoetes and in the adult lamprey accessibility rises to 80%. The results we have discussed show that at metamorphosis and during fasting the lysosomes undergo such changes as to determine an actual intracellular release of the acid hydrolases studied.