Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.     

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.     

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.     

Biocatalytic potential of lipase from Staphylococcus sp. MS1 for transesterification of jatropha oil into fatty acid methyl esters

An extracellular lipase producing isolate Staphylococcus sp. MS1 was optimized for lipase production and its biocatalytic potential was assessed. Medium with tributyrin (0.25 %) and without any exogenous inorganic nitrogen source was found to be optimum for lipase production from Staphylococcus sp. MS1. The optimum pH and temperature for lipase production were found to be pH 7 and 37 °C respectively, showing lipase activity of 37.91 U. It showed good lipase production at pH 6–8. The lipase was found to be stable in organic solvents like hexane and petroleum ether, showing 98 and 88 % residual activity respectively. The biotransformation using the concentrated enzyme in petroleum ether resulted in the synthesis of fatty acid methyl esters like methyl oleate, methyl palmitate and methyl stearate. Thus, the lipase under study has got the potential to bring about transesterification of oils into methyl esters which can be exploited for various biotechnological applications.     

19F-nuclear magnetic resonance: measurements of [O2] and pH in biological systems.

19F-nuclear magnetic resonance (NMR) has been used to determine both intracellular pH and oxygen concentrations in cell suspensions. Oxygen concentrations in Paracoccus denitrificans and insulinoma cells, RINm5F, in the NMR probe can be monitored directly by 1/T1 measurements of perfluorotripropylamine (FTPA)/lecithin emulsion added to the suspensions. With FTPA oxygen monitoring, we investigated the relative aeration capabilities of two types of NMR chambers. Both normal and transformed eucaryotic cells can be maintained in either chamber for at least 1-2 h at cytocrits of up to 20-25%, with 30% oxygen saturation and cell viabilities of 90-95%. Similar concentrations of procaryotes were maintained aerobic with high FTPA concentrations in the more efficient of the two NMR chambers. A new precursor molecule for the 19F-NMR pH indicator difluoromethylalanine, the para-chlorophenyl ester, has been tested and used in RINm5F cells and P. denitrificans, neither of which hydrolyzes methyl esters.     

Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin

1. Esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid (alpha-N-toluene-p-sulphonyl-l-norarginine) have been synthesized and shown to be hydrolysed by bovine trypsin and thrombin. As substrates for these enzymes, they were better than esters of alpha-N-toluene-p-sulphonyl-l-homoarginine or of alpha-N-toluene-p-sulphonyl-l-ornithine but not as good as esters of alpha-N-toluene-p-sulphonyl-l-arginine. 2. With trypsin as catalyst, the methyl and propyl esters are hydrolysed at the same rate at high substrate concentrations and hence deacylation of the acyl-enzyme appears to be rate-determining. In the presence of thrombin, however, the methyl ester is hydrolysed much faster than the n-propyl ester. 3. The variation of k(0) with pH indicates that groups with pK((app.)) values of 7.05+/-0.02 and 6.53+/-0.02 must be dissociated in trypsin and thrombin respectively for hydrolysis to proceed. 4. Activation constants have been determined for the trypsin-catalysed hydrolysis of methyl gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyrate and have been compared with the corresponding constants for the hydrolysis of homologous substrates. 5. Cholate increases k(0) and decreases K(m); the effects are more pronounced with thrombin than with trypsin.     

Problems with the application of quin-2-AM to measuring cytoplasmic free calcium in plant cells

Abstract Attempts have been made to use the fluorescent calcium quin-2 to measure cytoplasmic free calcium in a variety of plant cells. Failure to measure intracellular fluorescence can be attributed to extracellular hydrolysis of the membrane-permeable ester quin-2-AM used to load the cells. Attempts to overcome this problem by long incubation times showed that the by-product of ester hydrolysis, formaldehyde, is inhibitory to cell growth. Thus, it seems that the applicability of the acetoxymethyl ester of quin-2 for cytoplasmic calcium measurements in plant cells is limited, though quin-2 could still be very useful in some cells, especially if more suitable esters or micro-injection are used.     

Investigation of the effect of different extracellular factors on the lipase production by <Emphasis Type="Italic">Yarrowia lipolityca</Emphasis> on the basis of a scale-down approach

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.     

Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.     

Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of increasing extracellular pH on protein synthesis and protein degradation in the perfused working rat heart.

Increasing the extracellular pH over the range pH 7.4-8.9 stimulated protein synthesis by about 60% in the rat heart preparation anterogradely perfused in vitro. Protein degradation was inhibited by this pH increase. The magnitudes of the effects at pH 8.9 on protein synthesis and degradation were similar to those of high concentrations of insulin. Cardiac outputs were increased, as were cardiac phosphocreatine contents, indicating that the alterations in extracellular pH did not adversely affect the physiological viability of the preparation. ATP contents were unaltered. The creatine kinase equilibrium was used to assess the magnitude of the change in intracellular pH induced by these treatments. The increase in intracellular pH was about 0.2 for a 1-unit increase in extracellular pH. Thus small changes in intracellular pH have dramatic effects on cardiac protein turnover.     

Use of salicylic acid to measure the apparent intracellular pH in the Ehrlich ascites-tumor cell and Escherichia coli.

The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli. The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H+ by the cells estimated from the changes of the medium pH. Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s. When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found. The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking difference were found in the inherent permeability of the membrane to H+ and in the changes in this parameter by lowering the temperature to 2 degrees C.     

The effect of acid--base changes on skeletal muscle twitch tension.

The effects of acid--base alterations produced by changing bicarbonate (metabolic type), carbon dioxide tension (respiratory type), or both bicarbonate and carbon dioxide tension (compensated type) on skeletal muscle twitch tension, intracellular pH, and intracellular potassium were studied in vitro. Hemidiaphragm muscles from normal rats and rats fed a potassium-deficient diet were used. Decreasing the extracellular pH by decreasing bicarbonate or increasing CO2 in the bathing fluid produced a decrease in intracellular pH, intracellular K+, and muscle twitch tension. However, at a constant extracellular pH, an increase in CO2 (compensated by an increase in bicarbonate) produced an increase in intracellular K+ and twitch tension in spite of a decrease in intracellular pH. The effect on twitch tension of the hemidiaphragms showed a rapid onset, was reversible, persisted until the buffer composition was changed, and was independent of synaptic transmission. It is concluded that the twitch tension of the skeletal muscle decrease with a decrease in intracellular K+. The muscle tension also decreases with an increase in the ratio of intracellular and extracellular H+ concentration. However, there is no consistent relationship between muscle tension and extracellular or intracellular pH. The muscle tension of the diaphragms taken from K+-deficient rats is more sensitive to variations in CO2, PH, and bicarbonate concentration of the medium than that of the control rat diaphragms.     

Pentacyclic triterpenes from Myrianthus liberecus

From the methylated trunk wood extracts of Myrianthus liberecus, six pentacyclic triterpenes have been isolated as their methyl esters. These included the known methyl benthamate, methyl euscaphate, methyl tormentate, methyl arjunolate, methyl 3-isoarjunolate and methyl 3β-O-(4″-O-methyl-E-coumaroyl)-arjunolate, a new triterpene derivative.     

Intra- and extracellular pH of the brain in vivo studied by 31P-NMR during hyper- and hypocapnia.

Studies were performed to determine the pH relationships among the extracellular, intracellular, and arterial blood compartments in the brain in vivo. Resolution of the extracellular monophosphate resonance peak from the intracellular peak in 31P nuclear magnetic resonance (NMR) spectra of sheep brain with the calvarium intact enabled pH measurement in these respective compartments. Sheep were then subjected to both hyper- and hypoventilation, which resulted in a wide range of arterial PCO2 and pH values. Linear regression analysis of pH in these compartments yielded slopes of 0.56 +/- 0.05 for extracellular pH (pHe) vs. arterial pH, 0.43 +/- 0.078 for intracellular pH (pHi) vs. pHe, and 0.23 +/- 0.056 for pHi vs. arterial pH. These data indicate that CO2 buffering capacity is different and decreases from the intracellular to extracellular to arterial blood compartments. Separation of the extracellular space from the vascular space may be a function of the blood-brain barrier, which contributes to the buffering capability of the extracellular compartment. A marked decrease in the pH gradient between the extracellular and intracellular space occurs during hypercarbia and may influence mechanisms of central respiratory control.     

Biocatalytic synthesis of ascorbyl esters and their biotechnological applications

Ascorbyl fatty acid esters act both as antioxidants and surfactants. These esters are obtained by acylation of vitamin C using different acyl donors in presence of chemical catalysts or lipases. Lipases have been used for this reaction as they show high regioselectivity and can be used under mild reaction conditions. Insolubility of hydrophilic ascorbic acid in non-polar solvents is the major obstacle during ascorbic acid esters synthesis. Different strategies have been invoked to address this problem viz. use of polar organic solvents, ionic liquids, and solid-phase condensation. Furthermore, to improve the yield of ascorbyl fatty acid esters, reactions were performed by (1) controlling water content in the reaction medium, (2) using vacuum to remove formed volatile side product, and (3) employing activated acyl donors (methyl, ethyl or vinyl esters of fatty acids). This mini-review offers a brief overview on lipase-catalyzed syntheses of vitamin C esters and their biotechnological applications. Also, wherever possible, technical viability, scope, and limitations of different methods are discussed.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Analysis of the relationship between the reactivity of synthetic substrates and thrombin inhibitors and their structure

Kinetics of thrombin- and trypsin-catalyzed hydrolysis of diphenylacetyl-L-arginine esters was studied at pH 8.5 and 25 degrees C, and the antithrombin activity of in vitro synthesized compounds was examined. The anticlotting activity of arylsulphonyl-L-arginine methyl esters appeared to be higher than that of the derivatives of diphenyl arginine. Relations were found connecting polar (delta) and steric (Es) characteristics of substituent (R) in R-C6H4-SO2-Arg-OCH3 esters with their antithrombin activity in vitro or with efficiency of their thrombin-catalyzed hydrolysis. This gives supplementary possibilities for synthesis of new substrates and more potent thrombin inhibitors.     

Extracellular pH modifies mitochondrial control of capacitative calcium entry in Jurkat cells

It was found that a collapse of the mitochondrial calcium buffering caused by the protonophoric uncoupler CCCP, antimycin A plus oligomycin, or the inhibitor of the mitochondrial Ca2+/Na+ exchanger led to a strong inhibition of thapsigargin-induced capacitative Ca2+ entry (CCE) into Jurkat cells suspended in a medium at pH 7.2. The effect of these inhibitors was markedly less significant at higher extracellular pH. Moreover, dysfunction of the mitochondrial calcium handling greatly decreased CCE sensitivity to extracellular Ca2+ when the pH of extracellular solution was 7.2 (apparent Kd toward extracellular Ca2+ rose from 2.3 +/- 0.6 mm in control cells to 11.0 +/- 1.7 mM in CCCP-treated cells) as compared with pH 7.8 (apparent Kd toward extracellular Ca2+ increased from 1.3 +/- 0.4 mM in control cells to 2.4 +/- 0.4 mM in uncoupler-treated cells). Changes in intracellular pH triggered by methylamine did not influence Ca2+ influx. This suggests that, in Jurkat cells, store-operated calcium channels sense extracellular pH change as a parameter that modifies their sensitivity to intracellular Ca2+. In contrast, in human osteosarcoma cells, changes in extracellular pH as well as mitochondrial uncoupling did not exert any inhibitory effects on CCE.     

pH homeostasis in promyelocytic leukemic HL60 cells

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.