Availability of monovalent and divalent cations within intact chloroplasts for the action of ionophores nigericin and A23187

1. A23187 will uncouple electron transport by broken chloroplasts in a divalent cation dependent manner provided that they have been treated with a low concentration of EDTA.2. A23187 stimulates oxaloacetate-dependent oxygen evolution and inhibits phosphoglycerate reduction by intact chloroplasts isolated in a cation-free medium whereas the full effect of nigericin was dependent on the presence of external K⁺.3. Uncoupling of oxaloacetate reduction by A23187 in intact chloroplasts is inhibited by EDTA and this effect is overcome by excess Mg²⁺.4. The results suggest that divalent and not monovalent cations are available for collapsing the light-induced H⁺ gradient within the intact organelle.     

Rupture of rat liver lysosomes mediated by l-amino acid esters

Treatment of rat liver lysosome suspensions with 0.5–20 mM α-l-amino acid esters results in a progressive loss of latency of lysosomal enzyme activity. The increase in available acid phosphatase activity in lysosomal suspensions is correlated with the decrease of turbidity of these suspensions. Ester mediated turbidity decrease is dependent upon ester concentration, and the pH and ionic strength of the suspending medium. d-Stereoisomers of amino acid esters do not exhibit comparable capacity to damage lysosomes.α-l-Amino acid esters were found to be substrates for neutral lysosomal esterase and transpeptidase activity. The d-stereoisomers are degraded at much lower rates. These data support the hypothesis that ester dependent lysosomal rupture is mediated by the specific interaction of the ester with a structural or functional lysosomal protein.     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels.From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Kinetics of trypsin-catalyzed hydrolysis of some arginine-containing peptide methyl esters

The kinetics of trypsin-catalyzed hydrolysis (at pH 8.5) of methyl esters of some synthetic dipeptides containing the residues of both arginine and L(D)-p-fluorophenylalanine or L(D)-tyrosine has been studied. The digestion of Tos-(pF)Phe-Arg-OMe was shown as unfollowing the Michaelis-Menten kinetic since in the reaction course the substrate activation is observed and while the reaction product is the enzymatic process inhibitor. In contrast to this, the hydrolysis of other substrates studied, follows the normal Michaelis-Menten kinetic.     

Synthesis and hydrolysis of cholesteryl esters by isolated rat-liver lysosomes and cell-free extracts of human lung fibroblasts

The objective of this study was to examine and characterize the cholesteryl ester synthesizing [S] and hydrolyzing [H] properties of the acid cholesteryl ester hydrolase (acid cholesteryl ester hydrolase), both in isolated rat liver lysosomes and in cell-free extracts from cultured fibroblasts. For both liver lysosomes and fibroblasts extracts, the major synthesizing activity was found around pH 4 and did not require exogenous ATP. The rate of hydrolysis was measured at pH 4.5. Several different inhibitors were used in order to characterize the reactions. Ammonium chloride did not markedly affect the activity of acid cholesteryl ester hydrolase at pH 4 [S] or 4.5 [H], whereas chloroquine was a potent inhibitor of acid CEase in both liver lysosomes and fibroblast extracts. The [S] activity of the acid cholesteryl ester hydrolase in either material was not affected by the acylCoA:cholesterol acyltransferase inhibitor Compound 58-035 from Sandoz. Progesterone, on the other hand, which is an often used acylCoA:cholesterol acyltransferase inhibitor, markedly blocked both activities of the acid CEase. Our results indicate that the lysosomal compartment of both studied tissues, in addition to hydrolysis activity, also have a significant esterification activity. It appears that both activities are carried out by the same enzyme.     

Degradation of mucopolysaccharide in intact isolated lysosomes

The function of isolated lysosomes was studied by measuring mucopolysaccharide degradation. Cultured human diploid skin fibroblasts were grown in medium containing H235SO4 to label endogenous mucopolysaccharide. Lysosome containing preparations at various stages of purity were isolated from disrupted cells. These preparations degraded mucopolysaccharide as indicated by the release of radioactive sulfate. Degradation was temperature-dependent, required intact lysosomes, and was optimal when incubation was carried out at neutral pH in a buffer of low ionic strength. Lysosomes from Hurler fibroblasts were unable to carry out the degradative process. ATP at 0.5 mM was found to stimulate both the rate and the extent of mucopolysaccharide degradation; GTP, UTP, and CTP had similar effects, whereas the noncleavable ATP analog adenosine 5'-(beta gamma-imido)triphosphate gave no stimulation. The ATP stimulation was inhibited by nigericin. ATP also stimulated chloroquine accumulation in lysosomes, the magnitude of which was used to measure the change in intralysosomal pH. The presence of ATP was associated with acidification of lysosome pH by 0.23 units. Acetyl coenzyme A was also found to stimulate lysosome function. This reagent, however, had no effect on chloroquine accumulation and thus appears to stimulate mucopolysaccharide degradation by a mechanism different than that caused by ATP.     

Diacylglycerol hydrolysis in rat liver lysosomes

The matrix of rat liver lysosomes exhibits high hydrolytic activity towards 1,2-diacylglycerol with an optimum at pH 4.0. The lipolytic reaction follows Michaelis-Menten kinetics (apparent Vmax 470 nmol hydrolysed/min per mg protein; apparent Km 71 microM 1,2-dioleoylglycerol). Formation of 1- and 2-monooleoylglycerols indicates an initial attack at both the primary and secondary ester bonds. The lysosomal matrix also catalyses (re)acylation reactions, i.e. the formation of 1,2-diacylglycerol from 2-monoacylglycerol and free fatty acid. However, (re)acylation proceeds at a far lower rate than deacylation of diacylglycerol. Lysosomal diacylglycerol hydrolysis is sensitive towards non-ionic detergents, cationic amphiphilic drugs and the lipase inhibitor RHC 80267.     

The effect of dihydroxyacetone phosphate and 3-phosphoglycerate on O2 evolution and on the levels of ATP,ADP and Pi in isolated intact chloroplasts

Addition of dihydroxyacetone phosphate (2.5 mM) or 3-phosphoglycerate (2.5 mM) to a suspension of isolated intact chloroplasts, which contains P_i only in low concentrations (0.2 mM) leads to a competitive inhibition of P_i uptake in the light. In consequence, the ATP/ADP ratio is strongly decreased. The rate of O₂ evolution is also reduced under these conditions, but the degree of inhibition is much higher after addition of dihydroxyacetone phosphate than after addition of 3-phosphoglycerate. Therefore, besides the competitive inhibition of P_i uptake, additional effects of dihydroxyacetone phosphate and 3-phosphoglycerate on O₂ evolution and CO₂ fixation of isolated intact chloroplasts must occur, which are discussed.     

Long-acting contraceptive agents: in vitro hydrolysis of esters of norethisterone and levonorgestrel

The hydrolysis of 108 esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) was studied in vitro using a rabbit liver preparation. Introduction of a double or triple bond into a straight-chain ester did not inhibit hydrolysis but a marked reduction in hydrolysis was produced on replacement of a methylene group by an oxygen atom. Hydrolysis was inhibited by substituents at C2 of the ester chain except in short chain esters. Cyclopropylcarboxylate and cyclobutylcarboxylate were readily hydrolysed and introduction of a furan ring into the side-chain did not affect hydrolysis. No hydrolysis occurred with a cholesteryl carbonate ester or with a pentamethyldisilyloxy ether. Forty-nine esters of levonorgestrel (13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) were also studied. In general, the pattern of hydrolysis for these esters was similar to that of the norethisterone esters. However, with few exceptions the levonorgestrel esters were hydrolyzed more slowly. For those esters for which information regarding the biological activity was available, there was no correlation between the potency of the esters and their rate of hydrolysis in vitro.     

Diastereoselective hydrolysis of peptide esters by alkaline protease. Preparation of racemization-free peptides

A new and practical enzymatic procedure for preparative diastereoselective hydrolysis of peptide esters using the alkaline protease alcalase as a catalyst was developed. This procedure has been successfully applied to the resolution of peptide diastereomers and synthesis of racemization free peptides.     

Antibody-enhanced hydrolysis of steroid esters

Testosterone-1α-carboxyethyl thioether was conjugated through an ester bond with the fluorescent compound umbelliferone. Testosterone-1α-carboxyethyl thioether umbelliferone conjugate was devoid of fluorescence, but yielded a fluorescent product upon incubation with hog liver esterase or with the IgG fraction of a rabbit antiserum that binds 5α-dihydrotestosterone and testosterone with similar affinity (anti-dihydrotestosterone IgG). The fluorescent compound was obtained in solution after adsorbing the ester to anti-dihydrotestosterone IgG immobilized on agarose, indicating that the appearance of fluorescence was due to hydrolysis and not to formation of a stable ester antibody complex. The antibody-enhanced hydrolysis was pH and temperature dependent and was specific with respect to the nature of the steroid and the site of steroid-umbelliferone conjugation: normal rabbit IgG and IgG directed against heterologous steroids (e.g., cortisol or progesterone) were without effect, and anti-dihydrotestosterone IgG failed to promote the hydrolysis of testosterone-7-umbelliferone or of cortisol-21-umbelliferone esters. Moreover, the hydrolysis of testosterone-1α-carboxyethyl thioether umbelliferone conjugate by anti-dihydrotestosterone IgG was inhibited by the homologous hapten but not by unrelated steroids. Hydrolysis of steroid-umbelliferone conjugates was also promoted in a nonspecific manner by nucleophilic substances such as imidazole, tyrosine or cysteine, suggesting that the antibody effect may be due to the presence of nucleophilic residues at, or near, the combining site. The results indicate that antibody can have enzyme-like properties, but the turnover is low due to slow dissociation of the reaction product from the antibody. The quasi-esterase activity of anti-steroidal antibodies can be utilized for the development of an immunoassay for these hormones.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

Characterization of ascorbyl esters hydrolysis in fish

The hydrolysis of ascorbate mono-, tri- and polyphosphates by trout intestinal alkaline phosphatase was examined. K_m values were established as 1.19, 4.1 and 3.7 mM, respectively. The enzyme catalyzed ascorbate triphosphate hydrolysis with 60% efficiency of that for ascorbate monophosphate. With the K_m value of 1.19 mM for ascorbate monophosphate the trout enzyme exhibits similar affinity with this substrate as with p-nitrophenyl phosphate (1.00–1.67 mM). Two K_m values for micro- and millimolar ranges of ascorbate monophosphate concentrations ranges were calculated as: 27.9 μM and 1.19 mM, respectively. Specific intestinal alkaline phosphatase inhibitor L-phenylalanine (100 mM), inhibited reaction rate by 20% in 10 min. We conclude that alkaline phosphatase, which is in a great abundance in the trout intestine, serves as ascorbate esters hydrolase, thus releasing active ascorbic acid into circulation.