Sensitivity of lysosomal enzymes to the plant alkaloid sanguinarine: comparison with other SH-specific agents

The influence of the benzo[c]phenanthridine alkaloid sanguinarine on some lysosomal enzyme activities was investigated. Sanguinarine inhibits lysosomal hydrolases in homogenates of cultured mouse fibroblasts. After incubation of mouse fibroblasts in culture with 100 microM sanguinarine an approximately 50% decrease in the activities of N-acetyl-beta,D-glucosaminidase (NAGA), beta-galactosidase (GAL), arylsulfatase and acid lipase was observed. Because the biological activity of sanguinarine might arise from the interaction of its iminium cation with enzyme thiol groups, we compared its effect on NAGA, GAL and acid phosphatase (AcP) activities with the effects of SH-specific reagents p-chloromercuribenzoic acid (CPMA) and N-ethylmaleimide (NEM). Treatment of lysosomal fractions with millimolar concentrations of sanguinarine induces a dose-dependent inhibition of the enzymes; for example, 0.6 mM sanguinarine causes approximately a 40% decrease in AcP and NAGA activities. NEM has similar effects, and increasing the preincubation temperature from 0 degrees C to 37 degrees C intensifies the inhibition due to both agents. CPMA also inhibits the activity of GAL (IC50 0.7 microM), AcP (IC50 12.5 microM) and NAGA (IC50 6.8 microM) in a dose-dependent manner but is more potent than sanguinarine or NEM. Comparative analysis of the primary structures of these enzymes using the program BLAST reveals the presence of highly conserved cysteine residues, which confirms the importance of thiol-groups for their activities. Thus, both the experimental observations obtained in this study and the literature data imply a significant role of redox-based mechanisms in regulating lysosomal functional activity.     

The comparison of lysosomal enzymes activities in alveolar and peritoneal macrophages of rat

Studies were carried out on macrophages isolated from control and thioglycollate injected rats. Intraperitoneal injection of thioglycollate had no effect on the number of harvested alveolar macrophages but caused a marked increase in the number of peritoneal macrophages. The specific activities of all the investigated enzymes were significantly lower in peritoneal macrophages from control rats in comparison to alveolar macrophages. Thioglycollate stimulation of peritoneal macrophages caused increase in activities of all lysosomal hydrolases studied. Cathepsin B, N-acetylglucosaminidase and esterase showed the highest level of stimulation.     

Cholesterol esters in developing rat brain: enzymes of cholesterol ester metabolism

Abstract— Three enzymes of cholesterol ester metabolism, a cholesterol-esterifying enzyme which incorporates free fatty acids into cholesterol esters without participation of CoA, and two cholesterol ester hydrolases with differing pH optima, all showed distinct changes in developing rat brains. The specific activity of the esterifying enzyme was approx. 20 percent of the adult level at birth, increased gradually to the adult level by 20 days of age and remained constant thereafter. The pH 4.2 hydrolase at birth also had a specific activity of about 20 per cent of the adult level but it increased rapidly to reach a peak at 13 days, by which time the activity had increased eight-fold. The activity declined somewhat thereafter to reach the adult level by 23–30 days. In contrast, there already was 60 per cent of the adult specific activity of the pH 6.6 cholesterol ester hydrolase at birth. The activity remained constant until 12 days and then doubled during the next two weeks, reaching a broad peak, then declining slightly to reach the adult activity by 50 days. Therefore, the developmental changes of both of the hydrolases appeared to be related to the process of myelination. The period of active myelination (10–30 days) was characterized by the sharp rise in the activity of pH 6.6 cholesterol ester hydrolase and by the rapid decrease of pH 4.2 cholesterol ester hydrolase.     

The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain.

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.     

Lead-induced increase in antioxidant enzymes and lipid peroxidation products in developing rat brain

Pregnant rats were treated with 0.4% lead acetate through drinking water from 6th day of gestation and this treatment was continued till 21 post natal days (PND). Four regions of the brain namely hippocampus, cerebellum, frontal cortex and brain stem were dissected at 10, 20, 30 and 40 PND for estimation of lipid peroxidation products (LPP), catalase (CAT) and superoxide dismutase (SOD). The results indicate that there was a significant (P < 0.05) increase of LPP in exposed rats than their corresponding control at 10, 20 and 30 PND both in hippocampus and cerebellum. At PND 40, the LPP of control and exposed were found to be almost same in both the tissues indicating recovery from lead toxicity. CAT activity was significantly (P < 0.05) high in hippocampus of exposed rats up to PND 30 but up to PND 20 in cerebellum and frontal cortex. However, in brain stem, a significant (P < 0.05) increase in CAT activity was observed only at PND 10. A significant (P < 0.05) increase in SOD activity was observed up to PND 30 both in hippocampus and cerebellum on lead exposure. Frontal cortex exhibited a similar significant (P < 0.05) increase of SOD activity up to PND 20 and for brain stem up to PND 10. There was no significant change in the activity of antioxidant enzymes (CAT and SOD) and LPP in all the four brain tissues of control and exposed rats at PND 40 indicating recovery from lead-induced oxidative stress. This research work was presented as a poster in Annual Biomedical Research Conference for Minority Students (ABRCMS) at Dallas, Texas, USA, during November 10–13, 2004 and the abstract was printed on page 231 of the Conference Proceedings     

Turnover of myelin and other structural proteins in the developing rat brain

1. Protein metabolism of myelin and other subcellular components from developing rat brain was studied for periods from 5h to 210 days after intraperitoneal injection of [(3)H]lysine and [(14)C]glucose. 2. Half-lives for total brain proteins (t(0.5)) were 27 days after [(3)H]lysine and 4 days after [(14)C]glucose injection. 3. Factors accounting for the difference in the turnover rates obtained with different precursors, and the problem of reutilization of the label were investigated. 4. The catabolism of purified myelin proteins was studied and the half-lives of individual myelin proteins were calculated. 5. Myelin basic proteins turned over at two different rates. Half-life of the fast component of myelin basic proteins was 19-22 days and the slow component exhibited a high degree of metabolic stability. 6. Proteolipid protein underwent slow turnover. High-molecular-weight Wolfgram (1966) proteins underwent (relatively) fast metabolism (t(0.5) of 17-22 days).     

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

During contractions, when the rate of ATP hydrolysis exceeds that of ADP phosphorylation, inosine 5'-monophosphate (IMP) accumulates in skeletal muscle. If the cellular energy balance is not promptly restored, subsequent purine degradation to inosine via 5'-nucleotidase can occur, a process that is most robust in the slow-twitch red, as compared to fast-twitch, skeletal muscle. We measured the distribution of 5'-nucleotidase activity among membrane-bound and soluble fractions of fiber specific skeletal muscle sections and found most (80-90%) of the total 5'-nucleotidase activity to be membrane-bound. The 5' IMP nucleotidase activity present in the soluble fraction of muscle extracts differs among fiber types with slow-twitch red > fast-twitch red > mixed fibered > fast-twitch white. Experiments testing the substrate dependence of IMP and AMP dephosphorylation by the soluble fraction of muscle extracts revealed a lower Km toward IMP (approximately 0.7-1.5 mM) than AMP (1.9-2.8 mM). Among skeletal muscle fiber sections, the soluble 5'-nucleotidase activity present in slow-twitch red muscle extracts had the highest substrate affinity, the highest activity with IMP as substrate, and an estimated catalytic efficiency (Vmax/Km) that was > 3-fold higher than calculated for fast-twitch muscle extracts. This is likely due to the Mg2+ dependent cytosolic 5' IMP nucleotidase isoform, since immunoprecipitation experiments revealed 3-4 times more activity in slow-twitch red than in fast-twitch red or fast-twitch white fibers, respectively. These finding are consistent with the previously recognized in vivo pattern of nucleoside formation by muscle where the soleus demonstrated extensive inosine formation at a much lower IMP content than fast-twitch red or fast-twitch white muscle fiber sections.     

Phosphotransferases and lysosomal enzymes in fetal human and rat lung

The present investigations on rat lung show that metabolic changes occurring around the 20th gestational day are accompanied by multiple alterations in the quantitative pattern of enzymes. This involves increases in two lysosomal enzymes (N-acetyl beta-glucosaminidase and beta-galactosidase) and a rise and fall in pyruvate kinase and alpha-glucosidase. The striking transient upsurge of adenylate kinase, however, is postponed until after birth. The normal diminution of thymidine kinase and peptidylproline hydroxylase is drastically enhanced by an injection of cortisol to fetal rats. Studies on human pulmonary tissues consisted in determining enzyme concentration from the ninth to the 21st week of gestation and an histologically normal adult lungs. The results show that the 15th to the 21st week of gestation is the period of increase in pyruvate kinase, adenylate kinase and alpha-glucosidase. The rise during the development of several enzymes (e.g., 5'-nucleotidase, alkaline phosphatase, and gamma-glutamyl transpeptidase) and the decline in thymidine kinase and peptidylproline hydroxylase, however, dose not begin until after the 21st week of gestation.     

Platelets, lysosomal enzymes and anaphylactic shock in the rat

1. Rat serum levels in beta-glucuronidase and beta-galactosidase are higher than plasma levels. Rat platelets release these lysosomial enzymes during blood coagulation in vitro. 2. After anaphylactic shock, in the sensitized rat, there is no increase in beta-galactosidase and beta-glucuronidase plasma levels. The tissues of the sensitized rat do not release these enzymes during the antigen-antibody reaction. The blood platelet level is diminished after anaphylactic shock and the serum levels of the lysosomial enzymes are decreased. 3. In thrombopenic rat, anaphylactic shock is identical as in control animals. Rat platelets do not play a significant role in the anaphylactic shock.     

Exchange of sterols between myelin and other membranes of developing rat brain

1. The effect of inhibition of cholesterol synthesis by a hypocholesterolaemic drug (AY-9944) was studied in rat brain during development. 2. At 2 weeks after administration of AY-9944 to young rats 7-dehydrocholesterol accounted for half the total sterol of myelin and other subcellular components. 3. At 4 weeks after injection of the drug 7-dehydrocholesterol had disappeared whereas the cholesterol content of myelin had increased by an equivalent amount. Our studies show that purified myelin has low 7-dehydrocholesterol reductase activity and suggest that 7-dehydrocholesterol is largely converted into cholesterol outside the myelin sheath. 4. Resultant cholesterol may be re-incorporated into myelin by an exchange process. 5. The metabolism of sterols in developing brain is discussed.     

The urine from patients with peritonitis as a rich source for purifying human acid sphingomyelinase and other lysosomal enzymes

Milligram amounts of human acid sphingomyelinase (EC 3.1.4.12) were purified to 95% homogeneity using urine from patients with acute peritonitis. The activity of this enzyme is elevated more than 200-times in the urine of these patients. To a lesser extent, levels of some other lysosomal hydrolases are also elevated.     

Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta.     

In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver

Abstract— In vitro desaturation of [1-C¹⁴]linolenic, linoleic, oleic, and icosatrienoic acids was determined using homogenates and subcellular fractions of developing rat brain and liver. Linolenic, linoleic, and oleic acids were desaturated in the δ6-position and activity was optimal in the presence of CoA, ATP, MgCl₂, and NADH in a citrate-phosphate buffer at pH 6.0. Icosatrienoic acid was desaturated in the δ5-position with a much broader pH optimum. The unstable desaturation systems of brain were protected by reduced glutathione and niacinamide and markedly inhibited by dithiothreitol, p-chloromercuribenzoate, sodium cyanide or bathophenanthroline sulfonate. With brain homogenate of neonatal rats, the relative rates of desaturation of these substrates were 18:3(n - 3) > 18:2(n - 6) > 20:3(n - 6) > 18:l (n - 9). Specific activity of brain enzymes was greatest in neonatal rats with fluctuations in activity between 3 and 6 days of age. During this period, liver enzyme appeared to alter in a reciprocal manner. Total desaturation capacity of brain was maximal and fairly constant between 4 and 20 days of age, whereas liver activity increased dramatically after weaning. The activity of crude microsomal preparations from neonatal brain, like that of liver microsomes, was stimulated by a heat-labile component of the cytosolic fraction. These results demonstrate that brain has a high capacity for desaturation of the essential fatty acids during crucial stages of brain development when liver activity is relatively low.